the mitochondrial respiratory chain of yeast. structure and biosynthesis and the role in cellular...

Biochimica etBiophysicaActa, 895 (1987) 205-239 205 Elsevier

BBA86149

The mitochondrial respiratory chain of yeast. Structure and biosynthesis and the role in cellular metabolism

Simon de Vries and Carla A.M. Marres

Laboratory of Biochemistry and Section for Molecular Biology, Department of Molecular Cell Biology, University of Amsterdam, Amsterdam (The Netherlands)

(Received 12 April 1988)

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

II. N A D H dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 1. Identification of N A D H dehydrogenases in different growth conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 2. Structure and composition of purified N A D H dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

III. QH z : cytochrome c oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 1. Properties of the subunits containing prosthetic groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 2. Gene-disruption and gene-deletion studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 3. Biosynthesis and assembly of the bc I complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

IV. Cytochrome c oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

V. Lactate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 1. L-Lactate : eytochrome e oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 2. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

VI. Intermediary metabohsm and mitochondrial function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

VII. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Abbreviations: Q, QH 2, (ubi)quinone, (ubi)quinol; HQNO, 2-n-heptyl-4-hydroxyquinoline N-oxide; UHDBT, 5-n-undecyl-6-hy- droxy-4,7-dioxobenzothiazol; DTNB, 5,5'-dithiobis(2-nitrobenzoate); CD, circular dichroism; ENDOR, electron nuclear double resonance; ESEEM, electron spin echo envelope modulation; TMPD, N,N,N',N'-tetrarnethyl-p-phenylene diamine; DCIP, 2,6-di- chlorophenolindophenol; PAGE, polyacrylamide gel electrophoresis.

Correspondence: S. de "Cries, Universiteit van Amsterdam, Inter(sub)facultaire Vakgroep Biochemie, Afdeling Moleculaire Biologie/JHI, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.

0304-4173/88/$03.50 © 1988 Elsevier Science Pubfishers B.V. (Biomedical Division)

206

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

I. Introduction

The last review on the respiratory chain of yeast dates from 1973 [158]. It was devoted mainly to the peculiarities of the NADH dehydrogenase segment, in particular to question the presence or absence of site I phosphorylation. Shortly afterwards the research in this field shifted to the middle and terminal parts of the respiratory chain, a situation which still prevails. Consequently, our knowledge of the bc 1 complex and cytochrome c oxidase has increased considerably but compara- tively tittle progress has been made as to our understanding of the complicated NADH dehydrogenase.

The three large respiratory-chain enzymes, namely the N A D H : Q oxidoreductase, the QH2:cytochrome c oxidoreductase and the cytochrome c oxidase, were first purified from bovine-heart mitochondria in the early sixties. These enzymes have in common that electron transfer is coupled to the translocation of protons across the mitochondrial inner membrane. Their polypeptide composition is now fairly well established (25, 11 and 13 different subunits, respectively). In the 1970s, the first successful isolation of the bc 1 complex and cytochrome c oxidase from Saccharomyces cerevisiae and Neurospora

crassa were reported. In S. cerevisiae both enzymes contain nine different subunits. Only recently was the NADH : Q oxidoreductase from N. crassa purified and found to consist of some 22 polypeptides. In the 1980s, purified respiratory chain enzymes from prokaryotes became available. In all cases the subunit composition was found to be much simpler than that of their eukaryotic counterparts. Thus the bacterial bca

complex and the cytochrome c oxidase each contain three subunits (in some organisms the latter may even be a single-subunit enzyme) and the NADH : Q oxidoreductase contains only about ten different subunits [249]. The prokaryotic enzymes catalyse the same reactions as the eukaryotic enzymes and the composition and properties of the

prosthetic groups in the enzymes from both types of organisms are highly similar. Therefore, the catalytic mechanism of the respective enzymes from eukaryotes and prokaryotes are expected to be very similar, possibly even identical. It is clear that today's trend is towards the study of the simpler prokaryotic type of enzyme in order to understand the (minimal) structural requirements to couple electron transfer to proton translocation. At the same time, the study of the function of the subunits present only in the enzymes from lower and higher eukaryotes may reveal functions of the respiratory chain specific to more complicated organisms.

Simply because the respiratory-chain enzymes from bovine-heart mitochondria were for a long time the sole source of purified and abundant material, much more is known about the structure (i.e., size and shape and subunit contacts) and mechanism of action of these enzymes than of those from other sources. As to the mechanism, however, much has also been learned over the last decade from the bacterial enzymes, but less from the enzymes of yeast and fungi. With the advent of sophisticated immunological and molecular- cloning techniques, studies with yeast and fungi were greatly intensified. First, part of the mitochondrial DNA from yeast was sequenced so that the primary sequence of cytochrome b, of the three larger subunits of cytochrome c oxidase and of three (or two, depending on the organism) subunits of the ATPase became available [13,40,59,71,156,210]. Shortly afterwards the mitochondrial DNA from higher eukaryotes was completely sequenced and was shown to contain the same genes and seven additional open reading frames, recently identified to encode subunits belonging to the NADH:Q oxidoreductase [4,5,37]. Today, the primary structures, derived from the DNA sequence, of all nuclearly encoded subunits constituting the bc I complex and the cytochrome c oxidase from yeast are known. Furthermore, the sequence of almost all nuclearly encoded subunits of yeast cytochrome c oxidase was also de-

termined by protein sequencing. As to the nuclearly encoded subunits from the bovine-heart enzymes, so far their primary structure has been obtained only by sequencing the protein. Re- cently, the complete primary structures of the bacterial enzymes have been determined, mainly from sequencing the DNA. The three genes encoding the bc I complex are located on a single operon [44,46,75], the three genes encoding the cytochrome c oxidase of Paracoccus denitrificans are located on two separate loci containing additional unidentified open reading frames [179,200].

Comparison of the primary structure derived from the DNA sequence with that determined from the purified protein showed that the former predicts in a number of cases a polypeptide which is 20-80 amino-acid residues longer, extending from the NH2-terminus. This finding was of course in complete agreement with the original observation in pulse-chase labelling experiments that newly synthesized polypeptides are often longer than their mature counterparts (cf. Refs. 63, 95, 102, 138, 166, 190, 191 and 252). From these basic experiments a new research topic has emerged, mainly restricted to studies with yeast and fungi, dealing with the question how nuclearly encoded proteins synthesized in the cytoplasm and functional in the mitochondrion are transported towards this organelle (targetting, routing) and find their final destination (sorting) in one of the four mitochondrial compartments (inner and outer membrane, intermembrane space and matrix). A closely related subject is the study of the assembly of the respiratory chain complexes which are built from polypeptides encoded by both the mitochondrial and the nuclear DNA.

207

In this review we will discuss recent develop- ments concerning the structure, composition and assembly of respiratory-chain enzymes of yeast. Since our knowledge on the mechanism of action of these enzymes is derived mainly from studies with the enzymes from bovine-heart mitochondria and from prokaryotes, this topic is not reviewed and the reader is referred to the following papers: Refs. 11, 12, 24, 26, 27, 41, 53, 69, 101, 113, 175, 176, 178, 180, 199, 235 and 238-241. However, the mechanism of action of the L-lactate : cytochrome c oxidoreductase is reviewed because the enzyme is unique to yeast. Progress in the field of protein routing and sorting has recently been reviewed extensively [87,96,102,166,190,191,252]. In the second part of this review the role of mitochondria in the cellular metabolism of yeast grown under a variety of conditions is discussed.

II. NADH dehydrogenase

IL l . Identification of N A D H dehydrogenases in different growth conditions

It is well established that mitochondria from yeast, unlike those from mammalian cells, are capable of oxidizing externally added NADH. The oxidation of external NADH is not coupled to site I phosphorylation and is not inhibited by rotenone or piericidin, indicating that Complex I is not involved (cf. Table I). The enzyme responsible for the rotenone-insensitive oxidation of external NADH, and thus presumably of cytosofic NADH in vivo, is present in early log-phase cells of N. crassa [196] and Candida utilis (cf. Ref. 158), but

T A B L E I

O C C U R R E N C E O F V A R I O U S T Y P E S O F N A D H : Q 6 0 X I D O R E D U C T A S E A C T I V I T I E S I N M I T O C H O N D R I A F R O M

Y E A S T

log, l o g a r i t h m i c g r o w t h p h a s e ; s t a t , s t a t i o n a r y p h a s e ; n .d . , n o t d e t e r m i n e d .

T y p e o f N A D H Si te I R o t e n o n e Saccharomyces N. crassa o r C. utilis

d e h y d r o g e n a s e s e n s i t i v i t y l o g s t a t l o g s t a t

C o m p l e x I + + - - - +

I n t e r n a l + - - + - -

I n t e r n a l - - + n .d . + -

E x t e r n a l - - + + + -

208

disappears when the cells enter the stationary phase. In contrast, in S. cerevisiae this external NADH dehydrogenase is present both in the log phase and in the stationary phase [57,58,140,157]. The properties of the internal NADH dehydrogenase [39,88,230,251] involved in the oxidation of NADH produced in the matrix by the operation of the Krebs cycle, are dependent on the growth phase. Oxidation of intramitochondrial NADH- linked substrates by S. cerevisiae, C. utilis and N. crassa harvested in the log phase is insensitive to rotenone or piericidin and is not coupled to site I phosphorylation. Mitochondria from C. utilis and N. crassa harvested in the stationary phase possess site I phosphorylation, sensitive to the classic inhibitors of Complex I. The EPR spectrum of C. utilis mitochondria shows signals of Fe-S clusters closely resembling those of the Fe-S clusters 1-4 present in mitochondria or purified Complex I from bovine heart [2,9,39,88,159]. Surprisingly, only clusters 1 and 2 appear to be induced during the transition from log phase to stationary phase (or after release of iron or sulphur limitation), whereas clusters 3 and 4 are present under all conditions examined. Similarly, in P. denitrificans cluster 2 is absent in sulphate-limited cells [147]. It is not known whether the residual NADH-oxidase activity of sulphur-limited cells of C. utilis or P. denitrificans, which is not coupled to site I phosphorylation and which is insensitive to rotenone and piericidin, is due to a defective Complex I or to another type of internal NADH dehydrogenase.

In contrast to C. utilis and N. crassa, S. cerevisiae does not acquire rotenone-sensitive site I phosphorylation during the transition from log to stationary phase, or any other experimental condition tested so far (cf. Ref. 158). This is also true for the closely related yeast S. carlsbergensis. However, under conditions of starvation for carbon and nitrogen, Saccharomyces is capable of synthesizing an NADH dehydrogenase involved in the oxidation of intramitochondrial NADH that is coupled to site I phosphorylation but is insensitive to rotenone [140,157]. Since, in addition, the EPR spectrum of mitochondria of starved S. carlsbergensis did not show signals from Fe-S clusters, this type of internal NADH dehydrogenase is different from Complex I.

In addition to these dehydrogenases bound to

the mitochondrial inner membrane, yeast contains an outer-membrane NADH dehydrogenase. This enzyme is overexpressed in a ubiquinone-deficient mutant of S. cerevisiae [51]. In this mutant oxidation of external (cytoplasmic) NADH is not inhibited by antimycin, but only by cyanide. The P /O ratio was found to be the same as with ascorbate/TMPD. These and other observations indicate that cytochrome c is the electron acceptor of this outer-membrane electron-transfer chain (consisting of a flavoprotein NADH dehydrogenase and cytochrome bs) and that it shuttles electrons between the outer and the inner mitochondrial membranes.

II.2. Structure and composition of purified N A D H dehydrogenases

Recently the NADH dehydrogenase (complex I type) from N. crassa has been purified [107]. Similar to the enzyme present in bovine-heart mitochondria it contains up to 22 different subunits with molecular weights ranging between 10000 and 70000. It also contains Fe-S clusters and FMN as prosthetic groups. The specific activity of the purified enzyme is very low, which may have been caused by loss or inactivation of one or more polypeptides and/or by depletion of lipids during the purification.

In mammalian mitochondria seven subunits of the enzyme are encoded by mitochondrial DNA [36,37]. Likewise in N. crassa at least six subunits are of mitochondrial origin [17,52]. Significantly, these genes appear not to be present on the mitochondrial DNA of S. cerevisiae, which may explain why this yeast is not able to express a complex I type of NADH dehydrogenase [59,86]. The conservation of the primary structure of the mitochondrially encoded subunits of the NADH dehydrogenase is generally lower than for other mitochondrially encoded proteins of the respiratory chain. Although their role in the enzyme is at present unknown they are probably not directly involved in electron transfer (but possibly in proton translocation), since in none of these subunits sequence elements are present that are commonly found in Fe-S proteins.

The gross three-dimensional structure of the Complex I from N. erassa has been determined by

electron microscopy of membrane crystals [129]. Both in solution and in the membrane the enzyme is present as a monomer with a molecular weight of 610000. This finding seems at variance with ultracentrifuge experiments performed with the enzyme from bovine-heart mitochondria which was shown to be dimeric in solution [65]. In addition, kinetic studies indicate that the latter enzyme may be functionally dimeric [1,7]. The electron microscopy studies show that the Complex I from N. crassa is asymmetrically oriented with respect to the membrane. Approx. one-third of the mass is located in the membrane, a small part (approx. 5%) extends into one aqueous phase and the remainder into the other aqueous phase. Since the small mass is too small to contain the water-soluble flavoprotein segment (as identified in the Complex I from bovine heart and shown to contain the NADH-binding site [34,76,103]) and since Complex I catalyses the oxidation of intramitochondrial NADH, the large mass was proposed to be located on the matrix side of the inner membrane [129].

There are a few reports in the literature dealing with the purification of rotenone-insensitive NADH dehydrogenases from yeast. The purification of the external NADH dehydrogenases from S. cerevisiae and C. utilis has been described in two papers [47,141]. In both organisms the purified enzyme has a high molecular weight (1 500 000), but the subunit composition is simple, only two subunits in C. utilis (M r 250000 and 125 000) and but one (M r 250 000) in S. cerevisiae. In both enzymes the larger subunit was proposed to contain FMN as the prosthetic group. Later, it was indicated, however, that the enzyme isolated from S. cerevisiae is the cytosolic fatty acid synthetase [67]. Although in that work the conclusion as to the identity of the enzyme purified from C. utilis was less definitive, it is reasonable to assume that the enzyme purified from this organism is also the fatty acid synthetase, since the purification procedures used with both yeasts were the same.

In another study, the rotenone-insensitive internal NADH dehydrogenase was purified from C. utilis [139]. This enzyme consists of a single polypeptide of M r 37 000 and contains FAD. The K m for NADH was determined to be 16 ~tM. The

209

purified enzyme does not react with NADPH or lipoic acid, but can use DCIP, ferricyanide and Qa as electron acceptors. No information pertaining to the localisation of the NADH-substrate side of this enzyme with respect to the mitochondrial inner membrane was given, however. We have recently described the purification of a rotenone- insensitive NADH : Q6 oxidoreductase from mitochondria of S. cerevisiae [57,58]. The properties of this enzyme seem analogous to that from C. utilis referred to above, i.e., the enzyme consists of a single subunit (M r 53 000) containing one molecule of FAD per subunit (E m 7.6 = -370 mV), is specific for NADH (Km = 31 ~tM) and reacts with Q2, Q6 (the natural quinone in S. cerevisiae), Q10, menaquinone, DCIP and ferricyanide. The maximal turnover with Q2 as acceptor (2500/s) is about five times higher than with Q6. The enzyme contains only substoichiometric amounts of iron (0.08 mol Fe/mol FAD). The EPR spectrum of the purified enzyme shows no signals originating from the FAD- or Q6-semiquinone forms. Studies with polyclonal and monoclonal antibodies which inhibit the activity of the purified NADH dehydrogenase indicated that the NADH-oxidase activity in sonicated mitochondria (which in S. cerevisiae retain the right-side out orientation after sonication, in contrast to bovine-heart mitochondria [58]) was also inhibited by these antibodies, suggesting that the catalytic site of this enzyme is located at the outer face of the mitochondrial inner membrane. In addition, the K m values for the NADH-oxidase activity in mitochondria and the NADH:Q2 oxidoreductase activity in the purified enzyme were the same [58]. However, mitochondria isolated from a yeast strain in which the gene encoding this NADH dehydrogenase has been disrupted, showed no internal NADH- oxidase activity, indicating that the enzyme that was purified is the internal, rotenone-insensitive, NADH dehydrogenase (Marres, C.A.M., De Vries, S. and Grivell, L.A., unpublished observations). On the basis of the similarities between this enzyme and that purified from C. utilis we consider it likely that both enzymes are internal NADH dehydrogenases.

The purified NADH dehydrogenases from S. cerevisiae and C. utilis ar both specific for NADH. NADPH is not oxidized by either of these enzymes.

210

In our hands, mi tochondr ia or submitochondr ia l particles (i.e., sonicated mitochondria) f rom S.

cerevisiae did no t oxidize external ly added

N A D P H . There are reports, however, that soni-

cated mi tochondr ia from S. cerevisiae are capable

of oxidizing both N A D H and N A D P H and that

these activities are due to two dist inct (viz. depen-

dent on growth condit ions) N A D H and N A D P H

(Q69.) oxidoreductases [62]. In contrast , oxidat ion

of N A D P H in C. utilis via the respiratory chain,

l inked only to site II and site III phosphoryla t ion,

seems well established [20]. At present, we do not

know whether this species difference is real or whether under some condi t ions the N A D P H

oxidase activity is masked in S. cerevisiae.

III. QH2: cytoehrome c oxidoreductase

The QH2 :cy tochrome c oxidoreductase or b q complex, which catalyses the oxidat ion of QH 2 coupled to the electrogenic t ranslocat ion of pro-

tons across the membrane , has been purif ied from

m a n y sources. The enzyme from bovine heart mi tochondr ia is the most complex one conta in ing

11 subuni ts [142,144,231]; the bc I complex from S. cerevisiae or N. crassa conta ins 9 subuni ts

[116,142,225,235,236], the comparable enzyme

from chloroplasts or cyanobacteria , called the b6f complex, conta ins four subuni ts [101], and the

prokaryot ic bc 1 complex from P. denitrificans [123,250] consists of only three subunits . The

purif ied bc 1 complexes from Rhodobacter capsulata and Rhodobacter sphaeroides each conta in four

• polypeptides [133], bu t only three genes, arranged

in an operon, have been identif ied thus far

[44,46,75]. All these enzymes conta in the same n u m b e r and k ind of prosthet ic groups, namely a

cytochrome b carrying two hemes with different

redox midpo in t potentials , a cytochrome c l ( f ) and a high potent ia l [2Fe-2S] ferredoxin, the so-

called Rieske Fe-S protein. Moreover, the pr imary sequence of the polypept ides con ta in ing prosthetic groups is highly conserved in all species. Since the

mechan i sm of act ion of the Q H 2 : c y t o c h r o m e c

TABLE II

COMPARISON OF THE PROPERTIES OF THE SUBUNITS OF THE bq COMPLEX FROM YEAST AND BOVINE-HEART MITOCHONDRIA

M, methionine; aa, amino acids. 'Predicted helices' refers to the number of stretches of hydrophobic amino acids of sufficient length to span the lipid bilayer. Subunits of the yeast enzyme are listed according to their sequence on polyacrylamide SDS gelelectrophoresis.

Yeast Bovine heart

subunit M r length number of homology subunit M r number of PAGE a

presequence predicted (%) predicted (aa) helices helices

Core I 48225 b 17 0 core 1 49000 0 c 1 Core 2 38714 16 0 core 2 45 000 0 c 2 Cyt b 43633 - 8 51 Cyt b 42540 8 3/4 Cyt c 1 27419 61 1 56 Cyt c a 27221 1 4/3 Fe-S 20086 30 1 55 Fe-S 21536 1 5 17 kDa 14513 25 d 0 31 Hinge 9175 0 8 14 kDa 14430 M d 0 32 QPc 13 389 0 6 11 kDa 12269 M d 17 15 9.5 kDa 9507 0-1? 7

DCCD 7998 0-1? 9 7.2 kDa e 7262 M 1 27 7.2 kDa 7189 1 10

6.3 kDa 6363 1 11

a The numbers indicate the migration sequence on SDS gels according to Ref. 188. b Assuming a presequence of 17 residues. c Assumed to be the same as in yeast, since no sequence data are available yet. d Schmitt, M. and Trumpower, B.L., personal communication. e Sequenced by Philips, J. and Trumpower, B.L., personal communication. See text for further details.

oxidoreductases from all these different sources is best described in terms of a Q-cycle mechanism [41,53,151,199] and since the simplest bc 1 complex consists of only the three subunits that contain the prosthetic groups, these three polypeptides may be regarded as the catalytic core of the mitochondrial bca complex.

The primary sequence of the nine subunits present in the bc I complex from S. cerevisiae has been determined by cloning and sequencing the respective genes [8,50,135,145,156,161,185,217, 223,224,226] (see Table II). All subunits except cytochrome b are encoded by the nucleus. The sequence of the subunits of the bc 1 complex from bovine-heart mitochondria, except for the two core proteins, is also known and has been derived mainly by sequencing the purified peptides [14, 15,187 189,232-234]. A comparison of the primary sequence of the polypeptides of the bc a complex of the two organisms shows that all subunits of the yeast enzyme have homologous counterparts in the enzyme from bovine heart and that the latter contains two polypeptides not found in yeast. Homology is greatest between the subunits carrying prosthetic groups. The homology between the 11 kDa protein from yeast and the 9.5 kDa protein from bovine heart is very low, but nevertheless statistically significant [135]. Cytochrome b is the most hydrophobic subunit containing eight ([42,60,61], see below) putative transmembrane a- helices [186,237]. Cytochrome c a [183,185,232], the Rieske Fe-S protein [8,97,132,167,189,201] and possibly the 7.2 kDa protein [187,231] each contains one hydrophobic stretch of amino acids of sufficient length to span the membrane (see also below). The other polypeptides of the yeast enzyme are rather hydrophilic and are probably not anchored to the membrane via a transmembrane a-helix [50,161,217,223]. Analysis of the secondary structure of the 11 kDa protein by means of the Chou-and-Fasman rules indicated that it might fold into a transmembrane ,8-barrel structure similar to the porins [135]. However, application of the Gamier algorithm predicts a protein consisting mainly of a-helices and containing fewer ,8-turns. The homologous 9.5 kDa protein was proposed to contain a hydrophobic and an amphipathic helix [15]. The 17 kDa protein from yeast, homologous

211

to the so-called hinge protein, contains a contigu- ous stretch of 25 acidic amino acid residues [223].

III .1. Properties of the subunits containing prosthetic groups

The gene coding for cytochrome c 1 has been cloned and sequenced from a variety of organisms including S. cerevisiae, R. capsulata, P. denitrificans and N. crassa [44,46,75,123,183,185]. The primary sequence of bovine-heart cytochrome c 1 has been determined from the purified protein [232]. The amino acid sequence of cytochrome c 1 is well conserved among all eukaryotes, but considerably less when the sequences of eukaryotes are compared with those of prokaryotes. In all cases a sequence CXXCH is observed (positions 40-44 in the mature peptide of yeast), characteris- tic of hemoproteins containing covalently bound protoporphyrin IX and histidine as a ligand to the iron (Fig. 1). Furthermore, a conserved methionine residue (position 164) is present that is most likely the sixth ligand to the heme iron. This methionine is absent from the functionally homologous cytochrome f of chloroplasts or cyanobacteria [242,243], for which it was indicated that in addition to histidine, a lysine residue serves as a ligand [84,198]. Replacement of methionine by lysine may explain why the midpoint potential of cytochrome f is some 70-120 mV higher than that of cytochrome ca owing to the higher electronega- tivity of nitrogen as a ligand compared to sulphur.

In all cytochrome Cl sequences a non-polar stretch of 15-20 amino acids is present near the carboxy end (positions 212-226), which may fold as a membrane spanning a-helix. Removal of the last 70 amino acids from the carboxy terminus renders the cleaved cytochrome c a from N. crassa water-soluble in a monomeric state [124]. The interaction sites of bovine heart cytochrome c 1 with horse-heart cytochrome c have been mapped in two negatively charged regions [19,202] (see Fig. 1). These regions are quite well conserved amongst the various cytochromes c a from eukaryotes and prokaryotes. Possible interaction sites of cytochrome c a with the Rieske Fe-S protein or cytochrome b have not yet been determined.

212

Cytochrome c 1

C C H 69 82 NH 2

, III I I 40-44 cqt. c

binding

cleavage

170-177 o~- helix 164

iII I I H 212-226

cqt. e binding

COOH

I

2Fe-2S protein

cleavage o~-helix

NH 2

, I I 24 41 I I

55 72

T'-H P ~ L

148-151

129-134 CPCH

CTHLGC COOH

I III I II I '

127 145 173

O~S G~S P ~ S

Fig. 1. Architecture of cytochrome c 1 and the Rieske Fe-S protein of S. cerevisiae. The two proteins are proposed to be anchored to the membrane by a hydrophobic a-helix. The cleavage site in cytochrome c 1 and the Fe-S protein rendering the homologous proteins from N. crassa water soluble are indicated by dashed fines. Cytochrome ca: cysteines-40 and -43 are covalently linked to the heine. His-44 and M164 are figands to the heine iron. The regions between amino-acid residues 69-82 and 170-177 are involved in the binding of cytochrome c. Fe-S protein: the regions CTHLGC and CPCH are involved in the binding of the 2Fe-2S cluster. Amino

acid changes by which the protein becomes thermolabile are indicated. See also text for further details.

Tim primary structure of the Rieske Fe-S protein from N. crassa [97], R. capsulata [44,46,75], P. denitrifieans [123], S. cerevisiae [8] and spinach chloroplasts [201] has been derived from the DNA sequence, that of bovine heart mitochondria from the purified polypeptide [189]. The architecture of the Fe-S protein from S. cerevisiae is shown in Fig. 1. Close to the amino terminus it contains a hydrophobic region, which is also present in the sequences of this protein from other sources, of sufficient length to span the membrane. Removal of this region by chymotrypsin treatment (in N. erassa) yields a fragment with M r 16 000, containing the Fe-S cluster, that, in contrast to the intact polypeptide is now water-soluble in a monomeric state [131]. The carboxy terminus, in which the single 2Fe-2S cluster resides, is very well conserved in all sequences known today. There are two other regions in the sequence that are well conserved (residues 48-62 and 74-95), but not in the Rieske Fe-S protein from spinach chloroplasts. It is not known what the role is of their structures in the function of the protein.

Before sequence data of the Rieske Fe-S protein became available, studies with the Rieske

protein from Thermus thermophilus indicated that the 2Fe-2S cluster is different from that in the plant ferredoxins [68]. From the amino acid composition it became clear that the two iron atoms are not coordinated to four cysteinyl-sulphur figands, but that two sulphur ligands are replaced by nitrogen ligands, presumably originating from histidine. In addition, the optical absorption, CD, EPR and Mrssbauer spectra of this protein differ significantly from that of plant ferredoxins [49,68,69]. Finally, the ENDOR and ESEEM spectra gave direct proof that at least one nitrogen atom serves as a ligand to the Fe of the cluster [38,208]. However, there is no direct biochemical evidence that the protein purified from membranes of T. thermophilus is indeed a Rieske Fe-S protein in the sense that it is structurally and functionally integrated in the bc 1 complex of this bacterium and, furthermore, the purified protein contains two (spectroscopically indistinguishable) 2Fe-2S clusters, in contrast to the Rieske Fe-S protein of mitochondria which contains only a single 2Fe-2S cluster. Nevertheless, the CD, EN- DOR and ESEEM spectra of the Rieske protein as present in the bc 1 complex from mitochondria

indicated that this 2Fe-2S cluster contains at least one nitrogenous ligand. It was further observed that the ENDOR spectra of the bc 1 complex reduced either by ascorbate or by sodium dithionite were, within experimental resolution, the same [208]. In the EPR spectrum the lineshape of the Rieske Fe-S cluster is different when reduced by ascorbate and by dithionite [55,197]. It was established that the change of the lineshape is due to the reduction of Q, bound in the vicinity of the cluster, becoming QH 2 when the redox potential in the system is lowered [55]. The apparent lack of effect in the ENDOR spectra indicates that the conformational change of the cluster seen with EPR is not due to a replacement of a sulphur ligand by a nitrogen ligand (or vice versa) (cf. Ref. 208).

When the primary sequences of several Rieske Fe-S proteins became available (cf. Fig. 1), it was clear that the arrangement of the cysteine residues is markedly different from those in the plant ferredoxins, especially in the sequence CPCH. It is presumably this histidine that is directly coordinated to the Fe atom. As in the case of cytochrome f, the presence of nitrogen instead of sulphur as a ligand may explain why the redox midpoint potentials of the Rieske Fe-S clusters (140-300 mV) are substantially higher than those of the plant ferredoxin 2Fe-2S clusters ( -400-0 mV). Furthermore, this histidine or that present in the sequence CTHLGC may be responsible for the pH dependency of the midpoint potential [68,69].

Mutants in the Rieske Fe-S protein displaying temperature sensitivity of growth on non-fermentable carbon sources have been prepared by random mutation of a plasmid harbouring the gene encoding the Rieske Fe-S cluster followed by transformation of a yeast strain in which the genomic copy of the gene was deleted [132]. Five transformants were characterized further and the mutations have been located (Fig. 1). None of these mutants grew at 37 o C. The thermostability of the b q complex of the mutant in which Pro-173 was replaced by Set was affected most. The amount of ~he be 1 complex (determined optically) present in the mutants (each grown at their permissive temperature) was in all cases close to that found in the strain complemented with a plasmid carrying the intact gene. The turnover numbers of the

213

mutant bc 1 complexes ranged between 77 and 26% of that of the wild type, the decrease being greatest in the mutations occurring at positions 127, 145 and 173, i.e. all in the well-conserved fragment containing the Fe-S cluster. The effect of the inhibitors antimycin, myxothiazol and UHDBT on the catalytic activity was also studied. The binding of antimycin was not affected in any of the mutants, whereas all mutants showed a slight resistance towards myxothiazol. The binding affinity of UHDBT in the strains carrying a mutation at positions 72 and 173 was similar to that of the wild type. It was reduced two- to three-fold for the mutations at positions 127 and 145, but greatly increased in the mutant in which a threonine 55 was replaced by isoleucine. This finding may be suggestive of a role of this threonine residue in the binding of Q [132], but in which way and how isoleucine would cause tighter binding of Q (by a general hydrophobic or steric effect?) remains to be elucidated. Furthermore, this threonine is replaced by a serine in the other Rieske Fe-S proteins functioning in mitochondrial bc 1 complexes and the homologous position in the protein of chloroplasts is probably occupied by glycine [189].

The amino acid sequence of cytochrome b from many organisms is known and has been derived mainly by sequencing of mitochondrial DNA (cf. Refs. 4, 5 and 156). The primary structure is well conserved amongst all species. The secondary structure has been predicted on the basis of the hydropathy profile which indicated the presence of nine long hydrophobic regions interrupted by relatively hydrophilic segments [186,237]. These hydrophobic regions are long enough to span the lipid bilayer in an a-helical conformation (Fig. 2). In addition, two pairs of conserved histidine residues, embedded in the membrane, were proposed as ligands to the two heme groups present in the polypeptide. Further, the two histidines that coordinate to the same Fe atom of a heme are located in two different a-helices (II and V). In each of these two a-helices the two histidine residues, coordinated to the two different hemes, are separated by 13 amino acid residues [186,237]. In this arrangement the two hemes are located on opposite sides of the membrane with the two heme Fe-atoms separated by about 2.0 nm. This config- uration of the two heroes is reminiscent of that

214

Cytochrome b (nine helices)

Diu( 17)i. = j NH 2 ILl /

Diu(3 I) []

P1 K R S

H R Y T K___/ m.___/

Myx(137)

My x(275) MyxC25%__ [] i x

R 0 K H P

H D D K S

I [] \ / I [ ] ---J Ant(Z2e) L Diu(225)

stigO 47) COOH

Cytochrome b (eight helices)

Stig(147)

Flmq Hgx(275) / I M y x ( 2 5 6 ) ~ B ' - x

H,x('37,1g k~ ~ ] OUT R y R O K H P

M K R H D D K S

oi~(31) [ ] \ / I [~ \ / I IN / I i - - J Ant(228) I [ ] Diu(225) ~ COOH

Diu(17) " NH 2

Fig. 2. Structure of cytochrome b from S. cerevisiae. The nine-helical model is from Refs. 186 and 237 and the eight-helical model from Refs. 42 and 60. Transmembrane spanning a-helices are represented by vertical rectangles. The histidine residues in helices II and V (above) or in helices II and IV (below) serve as ligands to the two heme groups. The boxed amino-acid residues confer inhibitor resistance upon replacement (cf. Refs. 60 and 61). The unboxed amino-acid residues mark the border of the hydrophilic phases and the membrane. Assuming that diuron/antimycin specifically interact with cytochrome b-562 and myxothiazol/stigmatellin with cytochrome b-566 the position of the two hemes with respect to the matrix (IN) and the intermembrane space (OUT) can be

assigned. Diu, dittron; Ant, antimycin; Myx, myxothiazol; Stig, stigmatellin.

or ig inal ly p r o p o s e d in the p ro tonmot ive Q cycle [151]. Accord ing to the Q cycle, the bc 1 complex conta ins Q-b ind ing domains on oppos i t e sides of the membrane , one in the vic ini ty of cy toch rome b-566 and the 2Fe-2S cluster, and one d o s e to cy tochrome b-562.

T o d a y there is ample exper imenta l evidence suppor t ing the Q-cycle hypothesis , a l though mos t

of the d a t a are also cons is ten t wi th the concep t of the semiqu inone cycle in which the bc 1 complex is cons idered to con ta in a single Q-b ind ing site [238,239]. F o r a d i scuss ion on the exper imenta l d i s t inc t ion be tween these two mode l s the r eade r is referred to Refs. 53, 56, 112, 152, 181 and 239. One of the key fea tures of the p a t h w a y of e lec t ron t ransfer in the bcl complex is tha t cy toch rome b is

reducible via two independent routes. This is concluded from experiments in which the reduction of cytochrome b was monitored in the absence of inhibitors, in the presence of either antimycin or myxothiazol and in the presence of both these inhibitors [54,55,229]. Only in the presence of both inhibitors, reduction of cytochrome b is blocked [199,229]. It is important to note that the kinetics of reduction of cytochrome b are markedly different depending on whether antimycin or myxothiazol is present which indicates two different electron transfer pathways to cytochrome b [54,55]. Furthermore, the binding of antimycin to the bc 1 complex is not dependent on the presence of myxothiazol (and vice versa) and both inhibi- tots may bind to the enzyme simultaneously. As- suming that antimycin and myxothiazol exert their inhibitory effect in the same way as inhibitors of the bacterial photochemical reaction center, i.e., by occupying the Q-binding site [149], the above- mentioned experimental results, in combination with the observations that antimycin specifically prevents the formation of one type of semiquinone bound to the bca complex [55,160] and myxothiazol prevents the formation of a second type of semiquinone [54], strongly suggest that the enzyme contains two separate Q-binding sites. There is, however, no direct experimental evidence concerning their location with respect to the membrane.

Many mutants specifically affecting cytochrome b have been described. In some of these mutants the polypeptide is completely absent, in others varying amounts of apo- and holocytochrome b are found, or the optical spectrum is changed (cf. Refs. 18, 35, 122 and 148). In addition, almost all mutations conferring resistance to inhibitors such as diuron, HQNO, antimycin, funiculosin, myxothiazol, mucidin or stigmatellin map in the mitochondrial gene encoding cytochrome b (cf. Refs. 60, 61 and 212). Recently, the exact position of a number of mutants showing reduced affinity for a particular inhibitor has been determined [60,61]. This is shown in Fig. 2. Resis- tance to diuron (and HQNO) is obtained when Ile-17, Asn-31 or Phe-225 are changed into Phe, Lys or Leu (or Ser), respectively. Antimycin resistance is conferred by changing Gly-37 or Lys-228 into Val or Met, respectively. Reduced affinity for myxothiazol occurs when Gly-137, Asn-256 or

215

Leu-275 are replaced by Arg, Tyr or Ser, respectively, and resistance to stigmatellin occurs when I1e-147 is replaced by Phe. It is seen that according to the folding model of cytochrome b proposed in Refs. 186 and 237 the reduced affinity for a particular inhibitor can be caused by changing amino acid residues that are located on either side of the membrane. Although it cannot be ruled out a priori that a replacement of an amino acid on one side of the membrane affects the structure of the polypeptide on the other side of the membrane, it seems more likely that the mutation occurs in the inhibitor binding site itself, as was found to be the case for mutations in the herbi- cide-binding site of Photosystem II [111]. On the basis of new calculations, it was argued that helix IV may be omitted, resulting in a change of the polarity of helices I-III with respect to helices IV-VIII [42,60] (see Fig. 2). In doing so, the mutations affecting the binding of diuron all map at the same side of the membrane, and similarly in the case of antimycin or myxothiazol. Moreover, the sites affecting the binding of diuron and antimycin, which specifically inhibit the oxidation of cytochrome b-562, all map on one side of the membrane, whereas the sites affecting the binding of myxothiazol or stigmatellin, which primarily inhibit the reduction of the Rieske Fe-S and consequently that of cytochrome b-566, are now at the same side, and on the side opposite to that of diuron and antimycin [60]. Consequently, this latter folding model predicts a location for the heme b-562 and heme b-566 with respect to the matrix and intermembrane space as given in Fig. 2.

In conclusion, this new folding model for cytochrome b is fully consistent with the original protonmotive Q cycle in which the bc 1 complex was proposed to contain two distinct Q-binding domains on opposite sides of the membrane, each domain being structurally and functionally associated with a heme-b group [151].

111.2. Gene-disruption and gene-deletion studies

Since the genes coding for the subunits of the bc 1 complex from S. cerevisiae have all been cloned, one can, in principle, establish the role of each subunit in the function of the enzyme sep-

216

a ra te ly b y s t udy ing the e f fec t o f d e l e t i o n o f a

p a r t i cu l a r gene o n the s t ruc tu re and ca t a ly t i c ac-

t iv i ty of t he de fec t ive complex . I t is c lea r t ha t

de l e t i on o f c y t o c h r o m e b, c y t o c h r o m e c I o r the

R i e s k e F e - S p r o t e i n wi l l y i d d a ca ta ly t i ca l ly inac -

t ive enzyme . In genera l , howeve r , de l e t i on o f (or

m u t a t i o n in) a s ingle subun i t appea r s a lso to af-

fect (viz. lower) t he levels of o t h e r subuni t s . F o r

example , t he a m o u n t o f E P R d e t e c t a b l e F e - S clus-

te r in c y t o c h r o m e b m u t a n t s c o n t a i n i n g d i f f e r e n t

a m o u n t s o f this c y t o c h r o m e , was f o u n d to be

p r o p o r t i o n a l l y co r r e l a t ed to the a m o u n t o f op t i -

ca l ly de t ec t ab l e c y t o c h r o m e b [31]. T h e r e d u c t i o n

in the levels o f the r e m a i n i n g subun i t s gene ra l l y

o b s e r v e d in m u t a n t s in w h i c h a p a r t i c u l a r g e n e

has b e e n inac t iva ted , is n o r m a l l y n o t d u e to a

dec reased ra te o f t r a n s c r i p t i o n o r t r ans l a t ion , b u t

is r a the r caused b y an a p p a r e n t i n c r e a s e d sens i t iv-

i ty t owards p r o t e o l y t i c d e g r a d a t i o n (cf. Refs . 50

a n d 155). The re fo r e , the ef fec ts seen a f t e r d e l e t i o n

o f a s ingle gene o f t en h a v e a p l e i o t r o p i c cha rac te r .

I n o r d e r to i n t e r p r e t the ef fec ts seen u p o n

de l e t i on o f a gene, t ak ing the f o r e g o i n g in to c o n -

s idera t ion , the fo l l owing r a t i o n a l e is used. I n t he

case w h e n the leve l o f a s u b u n i t is n o t a f f ec t ed by

w h a t e v e r de le t ion , i t is a s s u m e d tha t this s u b u n i t

is i n sens i t ive to p r o t e o l y t i c d e g r a d a t i o n , w h e t h e r

o r n o t i t ha s b e e n a s s e m b l e d i n t o a c o m p l e x .

W h e n a d e l e t i o n causes a p r o p o r t i o n a l dec rea se in

the leve l o f a l l o t h e r subun i t s ( excep t fo r those

insens i t ive to p ro teo lys i s ) , this m a y be t a k e n as

e v i d e n c e t h a t t hese a s s e m b l e i n t o a c o m p l e x (ca ta -

ly t ica l ly ac t ive o r i nac t ive ) in o r d e r to b e c o m e

res i s t an t t o w a r d s p r o t e o l y t i c d e g r a d a t i o n . T h e de-

c r eased a m o u n t o f a s s e m b l e d c o m p l e x may , in

such a case, be a d i r ec t c o n s e q u e n c e of t he dele-

t i on caus ing a less e f f i c i en t a s sembly . T h e l o w e r e d

levels m a y t h e n be d u e to p ro t eo ly s i s o f t he f rac-

t i on tha t is n o t a s sembled . S imi la r ly , a d e l e t i o n

m a y cause a dec l i ne in t he leve l o f s o m e subun i t s

a n d a c o m p l e t e loss o f o thers , pos s ib ly i n d i c a t i n g

tha t t hose p r e s e n t ( in m o r e o r less c o m p a r a b l e

a m o u n t s ) a re a s s e m b l e d i n t o a s t ab le s u b c o m p l e x .

F ina l ly , t he c o m p l e t e loss o f m a n y subun i t s m a y

sugges t t h a t n o n e o f t h e m a s s e m b l e in to a sub-

c o m p l e x as the resu l t o f a p a r t i c u l a r de le t ion .

T h e resul t s o f d e l e t i o n s tudies a re s u m m a r i z e d

in T a b l e I I I . I t is s een tha t d e l e t i o n o f co re 1 or

TABLE IIl

EFFECT OF DELETION OF A PARTICULAR GENE ENCODING A SUBUNIT OF THE bc I COMPLEX ON THE LEVELS OF THE REMAINING SUBUNITS

indicates a level 70-100% of the wild type as determined by Western blotting; +, 20-70% of the wild type level; a, less than 20% of the wild-type level detectable; 0, below detection level; nd, not determined; SA, specific activity; TN, turnover number. Data on core 1 and core 2 deletion mutants and on the level of the 7.2 kDa protein in the various other mutants are from P.J. Schoppink (personal communication, see also text). Data on the 7.2 kDa deletion mutant are from B.L. Trnmpower et al. (personal communication).

Amount present

Core 1 Core 2 Cyt b Cyt c 1 Fe-S 17 kDa 14 kDa 11 kDa 7.2 kDa SA TN

Core 1 - ~- + a z A = + + + 2--5% Core 2 = - A = a = + + + 5--10% Cyt b = = _ b ~ 0 nd A A nd 0 0 Cyt c 1 nd nd nd - nd nd nd nd nd 0 0 Fe-S nd nd + ~ + ° - nd nd nd nd 0 0 17 kDa ~ ~ ~ ~ ~ - ~ ~- nd = = 14 kDa = -- A - - A = - - A + 0 0

11 kDa ~- ~ A ~ A ~ A -- + 0 0 7.2 kDa = = ~ ~ ~ = = ~ - 0 0

17 kDa .d = ~ 0 a a -- a A 0 0 0

a The amount of holocytochrome b is 2-5% found in the wild type. b Levels in mutants in which a truncated form of cytochrome b is synthesized. c Estimated from optical spectra. d A double mutant with petite phenotype. See Ref. 195.

core 2 does not affect the level of cytochrome Cl, the 17 kDa protein or that of the remaining core protein. The levels of the 14 kDa, 11 kDa and 7.2 kDa subunits are somewhat reduced, whereas the amount of the Fe-S protein is strongly decreased (Ref. 194, see also Schoppink, P.J., unpublished results). Table III also shows that, independent of the type of deletion, the levels of the two core proteins are the same as those in the parental strain. According to the rationale outlined above, this indicates that the core proteins are insensitive to proteolytic degradation irrespective of whether they associate to form a subcomplex (cf. Ref. 235), or whether they assemble in a defective complex or do not assemble at all. Since both core proteins are hydrophilic and bound to the matrix side of the inner membrane when the bc 1 complex is assembled [115], but likely behave as soluble proteins in the matrix when assembly is prohibited, their apparent stability may be explained by the absence of matrix proteases other than those involved in the cleavage of presequences. The amount of holocytochrome b is strongly decreased in the core deletion mutants. In the core 1 mutant there is still a considerable amount of apocyto- chrome b (some 50% of the wild-type level) as estimated from Western blots, but only 2-5% of this is converted into holocytochrome b, as judged from spectrophotometric measurements. The level of holocytochrome b in the core 2 mutant is somewhat higher (5-10%) and corresponds to that found in Western blots [161,194]. The core 1 mutant does not grow on non-fermentable carbon sources, but the core 2 mutant does, albeit slowly. Mitochondria obtained from both mutants exhibit myxothiazol- and antimycin-sensitive QzH2 : cytochrome c oxidoreductase activity, about 2-5% and 5-10% of the wild-type level for the core 1 and core 2 mutant, respectively. This indicates that the turnover numbers of these defective complexes, based on the specific activity and the cytochrome b content of the mitochondria, are (nearly) the same as that of the bc 1 complex in the parental strain. Thus the core proteins are not necessary for catalytic activity or even assembly per se, but their presence apparently increases the efficiency of assembly.

Deletion of the 14 kDa subunit leads to a strongly reduced level of the 11 kDa protein and

217

vice versa [136,194] (see Table III). In both mutants the amount of the Rieske Fe-S protein and cytochrome b are strongly reduced. Conversely, in cytochrome b mutants the level of Fe-S protein, the 11 kDa and the 14 kDa proteins are strongly reduced [50]. It was explicitly shown that the rate of synthesis of the two latter subunits is not affected in these mutants [50] and that in 11 kDa deletion mutants the rate of synthesis of the 14 kDa subunit, the Fe-S protein and cytochrome b is the same as in the wild type [136]. The low level of the subunits in both mutants is thus explained by an increased rate of degradation. Taking these findings together, they suggest that the 11 kDa subunit and the 14 kDa subunit help to stabilise cytochrome b, possibly by forming a stable subcomplex, resistant to proteolytic degradation (cf. Ref. 136). The Rieske Fe-S protein may have a high affinity for this subcomplex, but presumably does not play an important role in its formation (stabilisation), since the amount of cytochrome b in mutants lacking the Fe-S protein is only reduced by about 50% [132] (unfortunately data on the levels of the 11 and 14 kDa proteins in these mutants are lacking at the moment). It is further shown in Table III that the levels of core 1, core 2 and cytochrome c 1 are not affected and that the amount of the 17 kDa protein is somewhat reduced in mutants lacking either the 11 kDa or the 14 kDa protein [194]. The remaining defective complex is catalytically inactive in both types of mutant, but whether this is due to the absence of either subunit or simply to the absence of holocytochrome b a n d / o r the Fe-S protein cannot be decided.

Mutants lacking the gene coding for the 17 kDa subunit grow both on the fermentable and non- fermentable carbon sources at rates indistinguishable from the parental strain [195]. Analy- sis of the mitochondria of this deletion mutant by Western blotting and by spectrophotometry showed that all subunits were present at wild type levels. Initial studies indicated that the Q2H2 : cytochrome c oxidoreductase activity in mitochondria containing the defective bc 1 complex is about 40% of that in mitochondria of the parental strain [195]. Later it was established that under optimal conditions of pH and ionic strength, and employing yeast cytochrome c as the electron accep-

218

tor, the maximal turnover of the mutant bc I complex was, within experimental error, the same as that of the wild type (Schoppink, P.J. and Hemrika, W., personal communication). The mutant and wild-type mitochondria exhibited the same P / O ratios (both 1.8) for succinate or N A D H as substrate. One has to conclude from all these findings that the 17 kDa protein in yeast is not required for the assembly of the bc I complex, nor it is important for electron-transfer activity coupled to proton translocation [195]. This is at variance with the results obtained in studies in vitro which indicated that the hinge protein from bovine heart mitochondria, which is homologous to the 17 kDa protein from yeast, is indispensable in the formation of a complex between cytochrome c I and cytochrome c [118,119].

Studies in which the 17 kDa protein was deleted by the one-step gene disruption method further showed that in about 25% of the cases deletion of the 17 kDa gene was accompanied by an additional mutation (presumably in mitochondrial DNA) yielding strain with a petite phenotype (17 kDa- ' * ) [195]. Table III indicates that the level of the core proteins is not affected, whereas all other subunits, including cytochrome Cl, are present at strongly reduced levels. Cytochrome b and the 7.2 kDa subunit are completely absent. When a plasmid carrying the gene coding for the 17 kDa protein was introduced in this mutant, the wild- type phenotype was not restored, only the amount of cytochrome c 1 returned to the wild-type level. This suggests that cytochrome c 1 is stabilised in the presence of the hinge protein under conditions that the formation of a bq complex is somehow prevented. In contrast, when all other subunits are present at (near) normal levels, they can apparently take over the role of the 17 kDa protein in this [195]. What is called stabilisation of cytochrome c 1 in these two cases may actually be proper integration in the membrane or binding to a partially completed bq complex.

The effect of deletion of the Rieske Fe-S, cytochrome c I or the 7.2 kDa protein on the level of the other subunits has not been described in detail. Deletion of the 7.2 kDa subunit does not affect growth on glucose or galactose, whereas the growth rate on ethanol is greatly reduced. The amount of the other subunits is hardly affected

but the enzyme lacking only the 7.2 kDa subunit is catalytically inactive (Trumpower, B.L. et al., unpublished data). From the effect of deletion of other subunits on the level of the 7.2 kDa subunit (Schoppink, P.J., personal communication; see Ta- ble III) one may speculate that this protein is associated with the hinge protein and cytochrome cl, consistent with studies of the bovine-heart enzyme in which it was shown that upon cleavage of the bq complex, the corresponding subunit of the bovine-heart enzyme copurifies with cytochrome c 1 and the hinge protein. Thus it seems that cytochrome c 1 'stabilises' the 7.2 kDa protein, but that the latter is not required to stabilise cytochrome c 1.

III.3. Biosynthesis and assembly of the bc 1 complex

Table II indicates that core 1, core 2, cytochrome q , the Rieske Fe-S protein and the 17 kDa protein are synthesised as precursors, while the three smallest subunits lack a presequence, only their amino-terminal methionine residue being removed. The prepeptides of core 1, core 2 and presumably of the 17 kDa protein are removed in a single cleavage step [207,225], whereas the leaders of the Fe-S protein and cytochrome c 1 are cleaved off in two stages [77,98-100,185,222, 227]. This suggests that the various subunits may follow different routes during their import into the mitochondrion. No information is at present available on the mechanism of import of core 1, the 17 kDa, the 14 kDa, the 11 kDa and the 7.2 kDa proteins. The core 2 protein is transported in an energy-dependent manner into the matrix where the presequence is cleaved off. Its mechanism of import is probably similar to that of other proteins, synthesized as precursors, that function in the matrix (cf. Refs. 6, 87, 102, 145, 154, 166, 191, 192, 226 and 252). The core I may follow a similar route. Translocation of the Rieske Fe-S protein and cytochrome c 1 into the mitochondrion requires a membrane potential and occurs via translocation contact sites (presumably with the aid of a receptor protein) (cf. Ref. 193). The Fe-S protein is completely transported into the matrix where it is cleaved for the first time and then exported in an energy-independent manner to the outer face of the inner membrane. Subsequently, the protein,

which at this stage is membrane-bound, is cleaved for the second time, again by a matrix protease. In contrast to the Fe-S protein, cytochrome c 1 remains bound to the inner membrane after transit- cation across the two mitochondrial membranes, although there is uncertainty as to whether, at this stage, the (bulk of the) protein faces the intermembrane space or the matrix (see Refs. 99, 185 and 227). At this stage the protein is cleaved by a matrix protease, exported to the outer face of the inner membrane, a step requiting a membrane potential, and cleaved for the second time by a

219

protease residing in the intermembrane space [99]. This second sorting process is dependent on the presence of heme [77], suggesting that at this stage the prosthetic group is covalently linked to the intermediary-sized polypeptide.

With the newly acquired knowledge detailed above we have worked out a scheme indicating how the bc I complex might be constructed from its individual components (Fig. 3). For simplicity we distinguish four stages, which are however, not necessarily as distinct as suggested here: (1) transcription/translation and import, (2) insertion of

Assembly scheme for the mitochondrial bc I complex

I Synthes!s and import

D Matrix

IM

• IMS

OM

II Insertion of prosthetic groups

Ill Formation of subcomplexes

Matrix

IM

OM

Cytosol

IV Assembly of subcomplexes into mature complex

0M

Cytosol Cytosol

Fig. 3. Scheme showing a possible assembly route for the bq complex of S. cerevisiae. IM, inner membrane; OM, outer membrane; IMS, intermernbrane space. The relative sizes of the subunits and the hydrophobic membrane interior are to scale. Subunits are shown as the mature peptides, not as the precursors. The stalks of the Fe-S protein, cytochrome c 1 and the 7.2 (7) kDa protein represent their t ransmembrane spanning a-helices. Core 1, core 2, the Fe-S protein, cytochrome c 1 and possibly the 17 kDa protein are transported via translocation contact sites from the cytosol to the mitochondrial matrix. If the 17 kDa protein indeed follows this route, it would have to cross the inner mitochondrial membrane again in order to reach the in termembrane space. The 7.2 kDa, 11 kDa and the 14 kDa proteins are assumed to reach the in termembrane space simply by traversing the outer membrane. Insertion of

the prosthetic groups is assumed to take place in the matrix. See text for further details.

220

the prosthetic groups, (3) formation of specific subcomplexes and (4) assembly of subcomplexes into the mature complex.

In stage one, the nuclearly encoded subunits are synthesized in the cytoplasm and imported independently into the mitochondrion. Simulta- neously, cytochrome b is synthesized in the mitochondrion. Cytochrome b may 'sponta- neously' bind to the inner mitochondrial membrane, owing to its strongly hydrophobic character [130], possibly even during its synthesis. The core proteins and the Fe-S protein are transported into the matrix. Cytochrome c 1 remains bound to the inner mitochondrial membrane, and we assume that its heine-binding domain is located at the matrix side. The final localisation of the smaller subunits with respect to the mitochondrial inner membrane has not been determined directly and their mechanism of import is not known. It is reasonable to assume that the 17 kDa protein is located on the same side of the inner membrane as cytochrome c 1. The fact that the 17 kDa protein is synthesised as a precursor [223] suggests that import is energy-dependent, but the mechanism of import and sorting remains to be established. We propose that the 14 kDa, the 11 kDa and the 7.2 kDa subunits all three face the intermembrane space and that they reach this compartment simply by crossing the outer mitochondrial membrane (cf. Ref. 87). Studies on the localisation of the 14 kDa protein indicated that an antibody raised against this subunit partially inhibited the activity of the bq complex in sonicated mitochondria, but not in mitoplasts [110]. The authors concluded that the 14 kDa protein is therefore located on the matrix side, but since mitochondria of S. cerevisiae do not invert upon sonication [57] the interpreta- tion of these results is not straightforward. How- ever, it was also observed that the reduction of cytochrome b in the presence of antimycin, a pathway involving the Rieske Fe-S protein which is located at the outer face of the mitochondrial inner membrane, was inhibited by this antibody, whereas reduction of cytochrome b in the presence of myxothiazol, not requiring the Fe-S protein, was not inhibited [110]. The latter data suggest that the 14 kDa protein, like the Fe-S protein, faces the intermembrane space. Studies in which the bc 1 complex from bovine heart was treated

with the - S H reagent DTNB indicated that the activity of the enzyme was inhibited due to the fact that the Rieske Fe-S cluster was no longer reducible by substrate [143]. It was further concluded from the effect on the EPR spectrum of the Fe-S cluster that the binding of ubiquinone was prevented. The cysteine residue that, after modifi- cation, is responsible for these effects, is, however, not located on the Fe-S protein, but on the 9.5 kDa subunit [10], which is homologous to the 11 kDa protein from yeast. These findings support the view that the 11 kDa protein, like the Fe-S protein, faces the intermembrane space. Other, circumstantial, evidence in favour of such a location for all three subunits, is that they each lack a cleavable presequence.

In the second stage the prosthetic groups are inserted in the Rieske Fe-S protein, cytochrome b and cytochrome c 1. We assume that all three subunits receive their respective prosthetic groups from the matrix in reactions catalysed by a rhodanese-like protein and by heme lyases (but different from the heme lyase present in the intermembrane space catalysing the insertion of the heme in cytochrome c). After insertion of the prosthetic group the Rieske Fe-S protein is exported from the matrix to the intermembrane space [98,100], and is anchored to the mitochondrial inner membrane via a hydrophobic transmem- brahe spanning a-helix. Cytochrome c I is suggested to flip to the other side of the membrane after insertion of the heme group (cf. Ref. 99) and the peptide is then, for the second time, cleaved at the amino-terminal end, yielding the mature-sized protein. It is very well possible that other subunits belonging to the bq complex are involved in the insertion of prosthetic groups, for example core 1 and the 14 kDa and 11 kDa proteins may assist in the synthesis of holocytochrome b from apocy- tochrome b (see Table III). In the third stage various subcomplexes are formed. The mature subunits, which are up to this point randomly distributed in the matrix, intermembrane space or inner membrane, associate specifically and from this point assembly starts as an ordered process. Cytochrome b, the 11 kDa and the 14 kDa subunit (and possibly the Rieske Fe-S protein) form a stable subcomplex [136], which may prevent the loss of the two heme groups and /o r the Fe-S

cluster. Cytochrome q, the 17 kDa and the 7.2 kDa protein also form a subcomplex (cf. Refs. 117, 118, 195 and 231) and the two core proteins may associate with each other (cf. Ref. 235) before they bind to the matrix-facing side of the cytochrome b polypeptide. In the fourth stage, the various subcomplexes associate, yielding the catalytically active bq complex.

IV. Cytochrome c oxidase

The cytochrome c oxidase from yeast, previ- ously identified as a seven-subunit enzyme [177,184], was later shown to consist of nine different subunits [174]. The three largest subunits are encoded by the mitochondrial DNA and synthesised in the mitochondrion [13,40,70,211]. The six smaller subunits are encoded by nuclear DNA, synthesised in the cytoplasm and post- translationally imported into the mitochondrion (cf. Refs. 102 and 166). The three mitochondfially encoded subunits from the yeast cytochrome c oxidase are strikingly homologous to those synthesised by other mitochondria and to the three subunits constituting the cytochrome c oxidase of P. denitrificans [179,200]. Since the latter enzyme contains all the prosthetic groups present in the cytochrome c oxidase from eukaryotes and, likewise, catalyses electron transfer from ferrocyto- chrome c to oxygen coupled to the electrogenic translocation of protons across the membrane, the three mitochondfially synthesized subunits from the eukaryotic cytochrome c oxidases are generally believed to represent the catalytic core of the enzyme. Nevertheless, the cytochrome c oxidase from Thermus thermophilus HB8 consists of only a single subunit [69], has the same complement of prosthetic groups as the eukaryotic enzymes, and pumps protons. Since the primary sequence of this subunit is not known, the possibility exists that it contains sequence patterns otherwise present in subunits II and III, in particular since its subunit molecular weight amounts to about 75000, i.e., substantially larger than that of any subunit present in eukaryotic and other prokaryotic oxidases.

The remainder of this section specifically deals with the properties of the six nuclearly encoded subunits from the yeast enzyme. The reader is referred to recent reviews on the structure of the yeast and beef-heart enzyme for further informa-

221

tion [24-27,113,134]. Data on the mechanism of action, the structure and composition of the prosthetic groups and on proton pumping can be found in Refs. 11, 12, 69, 105, 175, 176 and 238-241.

The amino acid sequences of all nuclearly encoded subunits of the yeast enzyme have been determined by sequencing both the polypeptides and the DNA (except Cox7) [43,120,137,163,171- 174,215,247,248]. All subunits present in the enzyme from yeast have homologous counterparts in the enzyme of bovine-heart mitochondria [25], which itself contains four additional subunits (Via, b c and VIIb, see Table IV) [cf. Refs. 107 and 203]. Cox5a (and 5b), Cox7 and Cox9 show only weak homology to subunits IV, VIIa and VIII, respectively. In the case of Cox9, the similarity to subunit VIII hardly goes beyond the putative presence of a transmembrane a-helix. In addition to such a helix, Cox5a and subunit IV contain a short homologous hydrophilic domain whereas Cox7 and subunit VIIa show similar amino termini (cf. Ref. 25). As shown in Table IV, Cox4, 5a, 5b, 6 and 8 have a cleavable presequence required for targetting and routing the protein to the appropriate mitochondrial compartment [121,137, 150]. Cox7 and Cox9 do not contain a cleavable presequence, only the first methionine residue is removed [173,248]. Furthermore, Cox8 and Cox9 are cleaved at the carboxy terminus involving in both cases four amino acid residues [163,173]. The yeast Mn-superoxide dismutase, an enzyme located in the matrix, also lacks the last four amino acids as was concluded from a comparison of the amino acid sequence determined from the purified protein and that predicted from the DNA sequence [145]. Although it is possible that in all these cases the proteins are proteolytically degraded during isolation this seems unlikely especially in ~he case of cytochrome c oxidase, since a degradation of the other subunits would also be expected. Fur- thermore, in each case four residues are removed and with respect to cytochrome c oxidase the protease has a cathepsin B-like activity [173]. Con- cerning the Mn-superoxide dismutase another type of protease activity may be involved, The function of these carboxy-terminal tetrapeptides is unclear at the moment. They are not required for import (cf. Ref. 87).

222

TABLE IV

COMPARISON OF THE PROPERTIES OF THE SUBUNITS OF C Y T O C H R O M E c OXIDASE F R O M YEAST M I T O C H O N D R I A A N D BOVINE-HEART M I T O C H O N D R I A

M, methionine. Data on the percentage homology are f rom Ref. 25. In the text, we designate the subunits of the yeast cytochrome c oxidase with the nomenclature of the gene (for example: Cox5a means subunit Va from yeast), whereas for the enzyme from bovine heart, the Kadenbach nomenclature is used.

Gene Yeast Bovine

PAGE a M r N-terminus number of length of homology PAGE a

predicted presequence (%) helices (aa)

COX 1 I 56027 MVC 12 - 56 I COX 2 II 26 678 DVP 2 15 38 II COX 3 III 30 340 M T H 7 - 43 III COX 4 IV 14 570 Q Q K 0 25 24 Vb COX 5a Va 14858 AQT 1 20 14 IV COX 5b Vb 15 206 VQT 1 17 13 IV COX 6 VI 12627 SDA 0 40 28 Va

Via VIb VIc

COX 7 VII 6603 ANK 1 M 13 VIIa COX 9 VIIa 6303 AIA 1 M 17 VIII

VIlb COX 8 VIII 5 364 VHF 0 27 30 VIIc

a Sequence of polypeptide bands in gelelectrophoresis according to

Since Coxl and 2 contain the prosthetic groups (presumably cytochrome a3-CUb, cytochrome a in subunit I and Cua in subunit II) they are ab- solutely required to construct an active cytochrome c oxidase. In the bovine-heart enzyme proton pumping was inhibited completely by the action of D C C D which was shown to react with a glutamic acid residue present in subunit I I I [32]. The rate of electron transfer was reduced maximally 30% by this treatment. Later studies showed that removal of subunit I I I greatly decreased the proton-pumping activity of the enzyme without a concomitant loss of electron-transfer activity (cf. Ref. 241). The role of the smaller nucleafly encoded subunits in proton pumping or in electron transfer or in both is less well defined.

Null mutants in Cox4, 5a, 5b or 9 do not display cytoehrome c oxidase activity [43,66,215, 248], whereas a null mutant in Cox8 is fully active and the amount of enzyme formed in mitochondria is close to that found in the parental strain, i.e., Cox8 is required neither for activity nor for assembly [163]. In the case of null mutants in Cox4, the levels of the other small subunits were not

Power et al. [172] (yeast) and Kadenbach et al. [114] (bovine).

greatly affected whereas specifically the amount of Cox2, but presumably not its rate of synthesis, was decreased [66]. Treatment of yeast with a mutagen and selecting for the formation of an inactive cytochrome c oxidase indicated that the recessive nuclear mutants obtained in this way fall into at least 34 different complementat ion groups, implying that to synthesize an active nine-subunit cytochrome c oxidase in yeast at least 34 different nuclearly encoded gene products (and three mitochondrially encoded) are required [146,216]. These proteins may be involved in all sorts of 'nuclear-mitochondrial interactions' (gene expression, transcript processing, etc.), in receptor- mediated translocation and in the synthesis of heme a. Six of these complementat ion groups represent mutations in a structural gene leading to its complete absence or to the formation of a non-functional peptide. Analysis of these mutants showed that in the absence of Cox9 the levels of Cox2, Cox7 and Cox8 were greatly decreased, the effect on the level of other subunits being minor [146]. Mutants in Cox4 and Cox6 show residual cytochrome c oxidase activity (8 and 7% of the

activity found in wild-type mitochondria, respectively). In both cases aberrant peptides are formed migrating slower on SDS-PAGE, but no band is seen at the position of the wild-type peptide [121]. The residual activity may be due to the mutant peptide conferring some activity. Alternatively, Cox4 and Cox6 may not be required for enzymic activity but only for proper and efficient assembly (cf. Ref. 121), so that their absence causes a reduced level of cytochrome e oxidase, in which the turnover number is, however, not or hardly affected. Similar reasoning may apply to mutants in Cox5a or Cox5b. As to the latter, it was shown that the cytochrome c oxidase normally contains Cox5a, the level of Cox5b being very low [43,215]. It is not known under which conditions Cox5b comes to expression, but it was shown to be expressed to about 10-20% of the wild-type level in a strain carrying a mutation in the Cox5a gene. Introduction of the Cox5b gene present on a multicopy plasmid in the Cox5a mutant restored near wild-type levels of cytochrome c oxidase activity [215].

From the above it is clear that the assembly of cytochrome c oxidase is dependent on the presence of all subunits except Cox8. Assembly is also dependent on the presence of heme, specifically of heme a [33,83,153]. Apart from the fact that heme a is required to synthesize a proper Cox1, heme controls, at the transcriptional level, the synthesis of Cox5a and Cox7 [83,153]. Furthermore, intermediates of the biosynthesis of heme may inhibit the formation of an active enzyme. In N. crassa

pantothenate is required for assembly of cytochrome c oxidase, but not for the synthesis of the individual subunits [16]. The pantothenate is probably removed shortly before or directly after complete assembly of the complex. The mechanism of action of pantothenate is obscure, but it would be interesting to know whether this requirement is restricted to N. crassa or whether the enzymes from bovine heart and yeast also show this requirement.

V. Lactate dehydrogenase

Dependent on growth conditions several different types of mitochondrial lactate dehydrogenase are present in yeast. In general, these enzymes are

223

specific for either D- or L-lactate. NAD-linked D- and L-lactate dehydrogenase activities are induced when yeast is grown, either aerobically or anaerobically, at high (3%) but not at low (0.6%) concentrations of glucose, suggesting that their biosynthesis is subject to glucose induction [79]. However, induction of these enzymes may occur at low glucose concentrations under conditions in which mitochondrial respiration is inhibited (e.g., by antimycin or in m i t - mutants) or when mitochondrial protein synthesis is absent (e.g., in the presence of chloramphenicol or in r h o -

mutants) [70,79]. These NAD-linked lactate dehydrogenases have been suggested to reduce pyruvate to lactate, in vivo, thereby regenerating NAD from NADH, since the appearance of the two enzyme activities paralleled the appearance of L- and D-lactate in the medium and" because these lactate dehydrogenases are not present in mitochondria from cells grown on lactate or pyruvate or on lactate plus 0.6% glucose [79]. However, the formation of lactate compared to the formation of ethanol and glycerol to regener- ate NAD, is probably of minor importance in yeast (see also below).

Two other types of mitochondrial lactate dehydrogenases, unique to yeast, are induced when cells are grown aerobically on media containing lactate. Both enzymes are responsible, in vivo, for the oxidation of lactate to pyruvate. The electrons are not donated to NAD but to ferricytochrome c thus entering the respiratory chain at the level of site III. The D-lactate: cytochrome c oxidoreductase has been shown to contain FAD and Zn as prosthetic groups [90], the g-lactate : cytochrome c oxidoreductase is a flavohemoprotein containing cytochrome b 2 and F M N [115]. Each lactate dehydrogenase is induced only in the presence of its specific lactate isomer. Both enzymes are induced when cells are grown on a mixture of D- and L-lactate.

In contrast to non-fermentable carbon sources like ethanol and pyruvate for which all components of respiratory chain are required to promote growth of yeast, growth on lactate still occurs in the presence of antimycin in many yeast strains. The ATP produced solely by site III phosphorylation is sufficient for growth although the growth yield is reduced by a factor of about 11 compared

224

to that in the absence of antimycin [162]. In the presence of antimycin, the pyruvate formed by the action of the lactate : cytochrome c oxidoreductase is excreted directly or is transformed (partially) into ethanol a n d / o r is excreted in the form of Krebs cycle keto-acid intermediates. In the absence of antimycin about two-thirds of the pyruvate formed is oxidized via the Krebs cycle and the respiratory chain, the remaining one-third is probably used for biosynthetic processes.

Of the two lactate dehydrogenases discussed only the L-lactate:cytochrome c oxidoreductase has been studied thoroughly and we will discuss some of its properties below.

V. 1. L-Lactate ." cytochrome c oxidoreductase

The L-lactate : cytochrome c oxidoreductase or flavocytochrome b 2 has been purified in intact form both from S. cerevisiae and Hansenula

L-lactate: cytochrome c oxidoreductase

NN2 I K

8

Fcyt . b 2 I

43 56 138

HH 100

I I K

10:3

FMN 1 Activity

lactate I act ate : cyt. c ----I

f o r m cy t . c FIC 2:33 ( X ) ( X )

200 c $00 400 500

I I I I I COON intact 100 100 I I K K

296:328

NN2 I COOH

NH2 I

(O~p)4 42 .0 - -

(cleaved)

I COOH ( o~p' )4 4 . 0 4 . 2

NH2 I

c o r e lOO

NH2

X

I COOH ( × ~ ' ) 4 2 . 0 - -

£

NH 2 I I COOH ( ~ ' ) 4 0 15

NH2 I I I I COOH ( X~)4 0 73.7

Fig. 4. Structure of the intact and the various cleaved forms of the L-lactate: cytochrome c oxidoreductase and the effect of cleavage on the activity with cytochrome c or ferricyanide (FIC) as acceptor. Histidines 43 and 56 serve as ligands to the heme iron. The binding sites for lactate, cytochrome c and F M N are indicated. Lysines 8 and 103 have been determined as the cleavage points yielding the cytochrome b 2 core fragment. Cleavage at lysines 138, 296 and 338 was deduced from the amino acid sequence of the enzyme from S. cerevisiae combined with the estimates of the molecular weights of the various fragments as determined by

polyacrylamide gelelectrophoresis in the presence of SDS. Based on data from Refs. 80-82, 89, 108, 109, 213 and 214.

anomala [109,124]. The enzyme is a tetramer consisting of four identical subunits (M r 57 000) each carrying one heme group and one FMN. In older purification procedures from S. cerevisiae the purified enzyme was cleaved during the isolation by endogenous PMSF-sensitive proteases. In this type of preparation, which in addition to the intact enzyme has been used frequently for kinetic studies (see below), the polypeptide chain is cleaved near the NH2-terminus, removing seven residues and, in addition, at a position that yields one fragment with M r 36 000 (a), containing the cytochrome b 2 and one with M r 21000 (/3) (see Fig. 4) [109]. This cleaved enzyme is also tetrameric, the individual protomers being built from one 21000 and one 36000 subunit. The large fragment (M r 36 000) can be cleaved further yielding the so-called core (M r 11 000), containing the heme. This fragment has been sequenced and was shown to be homologous to the microsomal cytochrome b 5 [90,91].

Several different functional domains have been distinguished on the polypeptide by means of controlled proteolytic cleavage followed by mea- surement of the activity and binding of cytochrome c. The results of experiments performed with the enzyme from S. cerevisiae and H. anomala are combined and summarized in Fig. 4 [80-82]. It is seen that a dramatic loss of activity occurs when some 30 amino acids are removed from the/3-domain (becoming/3') [80,81]. The enzyme (af t ' ) is still in a tetrameric form even after removal of the core (yielding (X/3')4), which itself is released as a monomer [82]. The (e/3')4 is the smallest fragment containing flavin and considering that neither the a-chain itself which completely covers the e-domain nor the purified /3 or /3 ' fragments contain flavin, both e and/3" domains must be involved in the binding of this prosthetic group [80].

Br-pyruvate, an active site-directed inhibitor of the enzymic activity and analog of the substrate lactate, binds to Cys-233, located on the e-domain [3]. The (e/3')-domain and the slightly larger (Xfl ')-domain have very low lactate: cytochrome c or ferricyanide activities [80,108]. The uncleaved (Xfl')-fragment from H. anomala exhibits a high turnover, though only with ferricyanide [82]. The e-domain was shown to contain a high affinity binding site for cytochrome c as measured via the

225

binding of its Zn derivative [213,214]. These data indicate that the binding sites for lactate and cytochrome c are both located on the e-domain and that electron transfer from FMN to cytochrome c is specifically mediated by cytochrome b 2 .

Recently, the gene coding for the L-lactate : cytochrome c oxidoreductase from S. Cerevisiae has been isolated and sequenced [89]. The open reading frame encodes a protein of 591 amino acids (M r 65522). Comparison of the amino acid sequence derived from the DNA with that from the intact protein showed that the enzyme is synthesized as a precursor with an extension of 80 amino acids at its amino terminus. The mature protein thus consists of 511 residues (M r 56569). The hydropathy profile of the presequence shows similarities with that of cytochrome c a and, to a lesser extent, with that of cytochrome c peroxidase, i.e., it is very long and strongly basic and contains a long stretch of non-polar amino acid residues capable of spanning a lipid bilayer. It was shown that all three proteins are imported into the mitochondrion in a two-step process, which is probably mandatory for reaching their final destination, the intermembrane space [45,77,99,185, 227].

V.2. Mechanism of action

In the last decade the mechanism of action of the L-lactate : cytochrome c oxidoreductase has been studied intensively. The reaction pathway has been unravelled in great detail employing the stopped-flow technique, the freeze-quench technique in combination with EPR and by means of temperature-jump experiments in which the redox state of the cytochrome b 2 and flavin were monitored [30,206]. In the older experiments, the cleaved enzyme from S. cerevisiae and its (de- hemo) flavodehydrogenase derivative were used. In later work experiments were performed with the intact enzymes a n d / o r their deflavo or de- hemo derivatives from S. cerevisiae and H. anomala. The main difference between the cleaved and the intact enzyme is probably at the level of substrate binding and proton abstraction leading to differences in their respective turnover numbers and the K m for lactate (cf. Ref. 124). The rates of

226

the intramolecular electron transfer steps are not affected [170]. The electron-transfer pathway in the enzymes from the two different sources is essentially the same. There are slight differences in the values of the various kinetic constants and in the standard redox potentials of the prosthetic groups. Most important of these is probably that, whereas in H. anomala the values of Era, 7 of cytochrome b 2, of the F M N / F M N H 2 couple and the two associated one-electron redox couples are all very similar [204,205], the Era, 7 of the three flavin-redox couples in S. cerevisiae is lower than that of cytochrome b 2 by 50-60 mV as determined in equilibrium potentiometric titrations [30]. How- ever, in the pre-steady state these prosthetic groups were found to equilibrate rapidly and to behave as if they all have very similar standard redox potentials (see Table V) [30].

In the description and understanding of the pathway of electron transfer in the L-lactate : cytochrome c oxidoreductase one has to realize that each protomer of the tetrameric enzyme can accept three electrons in total, one in cytochrome b 2 and two in the ravin, whilst the substrate, L-lactate, is an obligatory two-electron donor [30]. Given the fact that the Era, 7 of the pyruvate/lactate redox couple ( - 190 mV) is much lower than that of any of the prosthetic groups in the enzyme, complete

reduction will be obtained by lactate (in equilibrium). Therefore, the kinetics of reduction of the prosthetic groups by lactate in the absence of an electron acceptor such as cytochrome c or ferricyanide are expected to be biphasic. Indeed, two phases of reduction are observed experimentally (see Fig. 5) [30]. Phase I comprises the rapid binding of four molecules of lactate to the tetrameric enzyme followed by a burst of small ampli- tude of reduction of FMN to F M N H 2 [28,30]. The subsequent monophasic rates of formation of F M N . H and reduction of cytochrome b 2 are the same. No lag in the reduction of any of the prosthetic groups is observed experimentally [30,170]. The ratio F M N . H/b~ + is, initially, close to unity. These events in which the electrons of one molecule of lactate are rapidly distributed over the prosthetic groups complete phase 1. A second 20-fold slower phase of reduction (phase II) is then observed in which the prosthetic groups become completely reduced. In this phase interprotomer electron transfer must occur, resulting in a redistribution of electrons yielding two molecules of oxidized FMN. This internal rearrangement may involve heme-heme, heme-flavin as well as ravin dismutation equilibria. In addition, conformational changes controlled by the redox state of the ravin a n d / o r cytochrome in one

TABLE V

PROPERTIES OF THE L-LACTATE : CYTOCHROME c OXIDOREDUCTASES FROM S. CEREVISIAE AND H. ANOMALA

S. cerevisiae H. anomala

Turnover 550 s - 1 1000 s - 1

K m (lactate) 0.6 mM 1.2 mM K d (Cyt c) n.d. 0.1/xM

Thermodynamic properties: redox midpoint potential (mV)

F-H F / F H F H / F H 2 F / F H 2 b2 F-H F / F H F H / F H 2 F / F H 2 b 2

eq. 0.35 - 4 7 - 5 7 - 5 2 kin. 0.52 - 2 0 + 20 0 eq.+pyr , nd nd nd nd

+ 6 0.38 - 2 3 - 4 5 - 3 4 - 1 9 + 6 0.54 - 19 - 6 3 - 4 1 - 2 5 nd 0.96 +71 - 133 - 31 - 2 2

K d for pyruvate (mM)

ravin oxidized 8 ravin half reduced 0.4 ravin reduced 15

n.d., not determined; eq. or kin: determined under equilibrium conditions or kinetically, respectively; pyr, pyruvate; F, FH, FH 2, oxidized, half-reduced and reduced ravin, respectively; F-H, tool flavosemiquinone per mol L-lactate:cytochrome c oxidoreductase. See text for details.

Reaction pathway of the L-lactate: cytochrome c oxidoreductase

227

lac la

1 la~J [] )

2 3

c(ox) c(red)'/~ Ipyr 4a

t°°f I--1 r-lI

I =z d F O l O q

JI a

Ml=q

4

pyr l- \

5

t-" I f iF.

c(ox) c(red) L l []

Fig. 5. Reaction pathway of the tetrameric L-lactate: cytochrome c oxidoreductase. Steps 1-3 mark the sequence of events occurring in Phase I, steps 4-6 that of Phase II. [] and I , oxidized and reduced cytochrome b2, respectively. % [] or [] and I , FMN, FMN. H and FMNH 2. In step 4, an internal rearrangement of electron occurs, yielding two (oxidized) FMN's per tetramer, thus allowing the complete reduction of the enzyme (step 6). Steps 1-3 and 4a-6a indicate the intermediates formed during steady-state turnover. Note that under these conditions maximally two electrons per monomeric unit are present. The exact pyruvate-dissociation step is

unknown. After Ref. 220. See also text.

protomer may modulate the rate of electron transfer in another protomer [169]. The rate of the second phase is, however, much lower than the rate of steady-state turnover. Therefore, the four protomers of the enzyme act independently under steady-state conditions with lactate as substrate and cytochrome c as electron acceptor (see Fig. 5) [28,169,206]. During steady-state turnover, each protomer acts as a two-electron donor/acceptor in which cytochrome b 2 is first reduced by FMNH 2 and, after oxidation by cytochrome c, by FMN- H. At pH 7 the rates of reduction of cytochrome b 2 by the ravin hydroquinone or flavosemiqninone are probably very similar, but at pH 6 reduction

by the flavin hydroquinone is 6-times faster. In fact this reaction, measured by temperature jump, is the fastest electron transfer in the enzyme re- corded so far [206].

The binding of cytochrome c to the L- lactate:cytochrome c oxidoreductase is governed by ionic strength. At low ionic strength a stable 1:1 complex is formed [213,214]. Likewise, the steady-state kinetics are dependent on the ionic strength, affecting the apparent K m for cytochrome c without an effect on Vm~ , [29]. At low ionic strength (and high concentration of enzyme and cytochrome c), the rate of the reaction is determined by the internal electron transfer from

228

flavin to cytochrome b 2. At high ionic strength the rate of the reaction is diffusion controlled. The electrical charge product of the reaction equals about - 6 indicating a collision between two re- actants of opposite charge [29]. All these features are comparable to those observed in the reactions of cytochrome c with cytochrome c oxidase, cytochrome ca or cytochrome c peroxidase.

Information on the binding and subsequent oxidation of lactate has been obtained using halogenated pyruvate compounds and [2- 3H]lactate. The proposed mechanism is shown in Fig. 6 and is based on the findings that in the presence of, e.g., bromopyruvate the [2-3H]lactate reduced enzyme catalyses both a transhydrogena- Lion reaction (yielding bromolactate in which the tritium is found in the C2 position and pyruvate) and a halide elimination (yielding pyruvate with the tritium on the C3 position and Br-) [3,218-220]. In these reactions, cytochrome b 2 remains reduced and, apparently, plays no role. The scheme indicates that the oxidation of lactate occurs via the carbanion intermediate and that the C2 hydrogen is bound transiently to the enzyme. The rate of hydrogen exchange between reduced

enzyme and solvent indicates the presence on the enzyme of a group with a pK of 15. Possibly an active-site base and/or the flavin N5 anion are involved in the binding of the proton [220].

It is evident from Table V that binding of pyruvate greatly affects the redox properties of the flavin, in particular leading to a stabilization of the flavosemiquinone [204,205]. Since the standard midpoint potential of the two-electron flavin redox couple remains more or less constant, the one-electron couple F M N / F M N . H becomes more oxidizing by the same amount that the FMN • H / F M N H 2 couples becomes more reducing. Even though both redox couples are sufficiently low in redox potential to reduce cytochrome c, the electron transfer involves the reduction of cytochrome b 2 as an obligatory intermediate Step. It is clear that reduction by FMNH 2 will occur, but that reduction by the flavosemiquinone is, thermo- dynamically, highly unfavourable in the presence of pyruvate. As a consequence, the rate of turnover of the enzyme is drastically decreased [204]. At high concentrations of pyruvate, the catalytic capacity of the L-lactate:cytochrome c oxidoreductase is only about 5-10% of the maximal

E( Fox ) I

+

B r - l a c t a t e

Fox B- it...

H I

Br-CH2-C-CO 2 OH

Fox BH

2

Br-c.r, -c0-2 OH

Fred BH

3

Br-CH2-C-CO- 2 0

E( Fre d ) 4

Br-pyruvate

I 5

Fox BH

+Br- CH2-CI-C0- 2

OH

I ,

E( Fox ) + pyruvate + H +

Fig. 6. Pathways of halide elimination (steps 5 and 6) and of intermolecular hydrogen transfer (steps 1-4) as catalysed by the L-lactate: cytochrome c oxidoreductase [219]. The active-site base ( B - ) is involved in the abstraction of a proton from (Br - ) lactate yielding the carbanion, the common intermediate in halide elimination and intermolecular hydrogen transfer. See also text for further

details. Fox and Fr~, FMN and FMNH2, respectively.

capacity. Such a redox control mechanism may be of physiological significance to regulate the activity of the enzyme in situ [204]. Although the mechanism by which the flavosemiquinone is stabilized by pyruvate is not clear, we suggest that interprotomer interactions leading to a conformational change in the enzyme may play a role in this, thereby rationalizing the fact that the enzyme is a tetramer.

VI. Intermediary metabolism and mitochondrial function

The metabolic versatility of yeast is illustrated by its capacity to grow on a wide variety of carbon and nitrogen sources. Yeast may use ammonia, nitrate or (most of the) amino acids as the sole nitrogen source for growth. The carbon sources can be classified in two groups, i.e., fermentable substrates (mono-, di-, tri- or polysaccharides) and non-fermentable substrates (ethanol, glycerol, lactate, pyruvate or acetate). Fermentable substrates may be metabolized anaerobically in which case ATP is derived solely from glycolysis, whereas growth on the non-fermentable sources requires the presence of oxygen (cf. Ref. 72). Nevertheless, yeast cannot grow in the complete absence of oxygen, since molecular oxygen is required for the synthesis of heme, ergosterol and unsaturated fatty acids (cf. Ref. 127). Addition of Tween 80 plus (ergo)sterol to the medium allows some yeasts (S. cerevisiae) to grow anaerobically. However, other yeasts like C. utilis do not grow under these conditions. Moreover, C, utilis does not grow aerobically in the presence of a respiratory chain inhibitor, or to put it differently, the petite phenotype is lethal (cf. Ref. 221). In general, there seems to be a correlation between the tolerance of the petite phenotype and the presence of the Crabtree effect (i.e., S. cerevisiae shows alcoholic fermentation in the presence of excess sugar and oxygen whereas C. utilis does not produce alcohol under these conditions), but the molecular basis for this correlation is not understood [221]. The petite mutation (or addition of a respiratory chain inhibitor) in combination with the absence of an active mitochondrial adenine-nucleotide carrier (either in a mutant or by addition of bonkrekic acid) under which condition the mitochondria are completely

229

depleted from ATP is, however, lethal to S. cerevisiae [78]. With respect to metabolism C. utilis and S. cerevisiae are the two most thoroughly characterized yeasts.

It is obvious from its versatility that a complete overview of the metabolism of yeast is not available at present. Under different growth conditions different metabolic pathways are operative. Under conditions of starvation, i.e., when no net biomass is formed, endogenous carbohydrates (glycogen and trehalose) are used, and via an unknown trigger and mechanism the de novo synthesis of some enzymes and the active proteolytic break- down of others is induced (cf. Refs. 72 and 228). Moreover, the particular enzymes involved in any of these processes are different in different yeasts. Some general statements can be put forward, however, which apply to all or most yeasts. Like bacteria, yeast expresses specific sets of enzymes dependent on the carbon source as is well docu- mented for growth on glucose, galactose, maltose, raffinose, lactate, glycerol and alcohol (cf. Refs. 72 and 73). When nitrate instead of ammonia is used as the nitrogen source the enzymes of the pentose-phosphate pathway are present at a three-fold higher level to furnish the cell with reducing equivalents in the form of NADPH [22]. During growth on glucose the uptake systems of other sugars are repressed (catabolite repression), most mitochondrial enzymes are present at very low levels as well as the enzymes involved in gluconeogenesis (except those active in glycolysis) (cf. Ref. 72). Expression or repression is generally regulated at the level of transcription and is furthermore, for some enzymes, dependent on the presence of heme, oxygen, 'actively respiring mitochondria', compounds from intermediary metabolism and other factors yet to be identified. For example, induction of ADHII and the enzymes of the gluconeogenic pathway does not occur in petites of S. cerevisiae or even in grand cells grown on minimal medium. As to the latter, these enzymes are induced when aspartate, glutamate, malate or fumarate are added to the minimal medium, but their inducible effect is an- nihilated by the simultaneous addition of oxaloacetate [244]. The metabolic and mechanistic bases underlying these phenomena are at present only scarcely known.

230

The metabolism of glucose by yeast, aerobically or anaerobically, is quite well understood in terms of the particular set of enzymes involved and the flow of reducing equivalents. In general, it may be stated that the N A D / N A D H and the NADP/NADPH redox couples play distinct roles in metabofism, the former being predominantly involved in catabolic processes, whereas the latter mainly supplies the reducing equivalents required for biosynthesis [20-24,125,126,128]. Since glucose is more reduced than biomass the amount of NADH liberated in catabolism exceeds the amount of NAPH utilized in anabolism resulting in a net production of reducing equivalents as quantita- tively expressed in the assimilation equation for anaerobic growth of S. cerevisiae on glucose and ammonia [20-24,221]:

737 C6H1206 +680 NH 3 + 6 H2SO 4 +931 N A D P H

+ 1349 NAD + ~ 1000 C4H7.3202.24N0.6880.oo 6 (100 g ceUs)

+424 CO 2 +931 N A D P + +

+ 1349 N A D H + 1358 H 2 0 + 418 H +

Although the net production of reducing equivalents amounts to 418 'H 2' per 100 g cells, the actual amount of reducing equivalents to be oxidized in order to sustain growth equals 1349 'H 2' in the form of NADH, if one assumes that the flow of reducing equivalents in catabolism and anabolism are separate. This proposal was tested as follows. The products formed from glucose during anaerobic growth are ethanol, glycerol and to a far lesser extent acetate, succinate, pyruvate [128,221]. The formation of ethanol and glycerol have a simple rationale, i.e., to maintain the cellular redox balance. The conversion of glucose into ethanol and CO 2 is redox neutral and yields ATP. The conversion of glucose into glycerol requires NADH and ATP. The NADH originates from the excess reducing equivalents produced in the formation of biomass. Importantly, the amount of glycerol produced corresponds to the reoxidation of 1349 NADH and not to 418 NADH, indicating that there is no net and/or direct transfer of reducing equivalents from NADH to NADP and vice versa [21]. This same conclusion could be drawn by analyzing the growth yield on mixtures

of glucose and formate (using either ammonia or nitrate as nitrogen source), i.e., the excess NADH produced in the oxidation of formate could not act as a source of NADPH [22,23]. Furthermore, it was estabfished that the pentose-phosphate pathway is the main route to supply the yeast cell with NADPH. When nitrate was used as nitrogen source instead of ammonia, in which case four NADPH are required to convert nitrate into ammonia, the activity of the glucose-6-phosphate dehydrogenase was increased threefold. In contrast, the activities of the isocitrate dehydrogenases (NAD-linked in the mitochondrion and NADP- linked in the cytosol) were not greatly affected. In addition, it was calculated that the contribution of the NADP-linked isocitrate dehydrogenase reaction is insufficient to meet the NADPH requirements for growth, in particular for growth on nitrate [21,22,221].

During aerobic growth on high concentrations of glucose, S. eerevisiae excretes less glycerol and ethanol than during anaerobic growth. Part of the excess NADH is now being reoxidized via the respiratory chain yielding about 30% of the total amount of ATP produced under these conditions [125[. The growth yields and the rates of glucose consumption both aerobically and anaerobically are, however, the same, indicating that a Pasteur effect is absent. The apparent absence of a Pasteur effect is related to the high, but near physiological glucose concentration used which causes catabo- rite repression [125,127]. In contrast, in glucose- limited chemostat the rate of glucose consumption is about five times higher anaerobically than aerobically, i.e., a Pasteur effect is observed. No fermentation products are, however, formed aerobically and the growth yield is about five times higher (cf. Ref. 72). Growth on other sugars, for example on galactose and maltose, is usually slower than on glucose. During growth on galactose most mitochondrial enzymes are largely dere- pressed and about 85% of the total amount of ATP is derived from the action of the respiratory chain. For growth on maltose in which case dere- pression is only partial 40% of the ATP is produced by the respiratory chain. The growth yield (g cells/mol sugar metabolized) for aerobic growth on galactose is twice that of growth, aerobically or anaerobically, on glucose [126]. Complete oxida-

231

tion of 1 mol galactose yields 26 mol ATP (site I phosphorylation is absent), instead of 2 mol ATP in fermentation. Since during aerobic growth on galactose 30% of the sugar is completely oxidized, the amount of ATP formed aerobically is 4.5 times higher than anaerobically. Clearly, the expenditure of ATP in processes not related to net biosynthesis is much greater for aerobic growth [126]. This is

even more pronounced for growth on ethanol. This ATP may be consumed for the maintenance of solute gradients or in futile cycles. Further- more, some metabolites (acetate or propionate) may simply act as uncouplers [210].

Growth on non-fermentable substrates requires metabolically active mitochondria and induction of the enzymes of the gluconeogenic pathway. For

NAD(P) NAD(P)H

.__X~__~ R-5-P

cytosol

GAP

T ADP, NAD

~ ATP~ NADH

PEP NN~T

ADP NAD(P) NAD(P)H

e glu o~-Og ATP

glycerol glucose

ADP G-6-P

acetaldehyde ~,) oc-Gly-P F-6-P

NAD J 11b 11. T ethanol QH 2 m NADH F-l,6-diP

DHAP ,

! H +

mitochandr ian r_ ~

l.co K o:.. '°,'/

.AD . A O .

13

ATP ADP i ~ , ~ "~/, oxaloecetate pyruvate s

OH- /, ." k~ . NAD(P)H,'"~6 /('+N

/NAO / citrate , malate

I ' Ae.CoA 1

so cioote \ I isoc,tro,e

i IDP ATP Ac.carn , Ac.CoA ~ %/ acetate

asp

3 NADPH ~>T2 NADP acetaldehyde NADH ~ NAD 1

ethanol

Fig. 7. Schematic overview of the various metabolic pathways involved during growth of yeast on different types of non-fermentable carbon sources. Enzymes: 1, alcohol dehydrogenase (ADHII); 2, aldehyde dehydrogenase; 3, acetyl-CoA synthetase; 4, L-lactate : cytochrome c oxidoreductase; 5, pyruvate carboxylase; 6, malic enzyme; 7, pyruvate kinase; 8, phosphoenolpyruvate carboxykinase; 9, fructose biphosphatase; 10, glycerol kinase; l la , a-glycerolphosphate dehydrogenase (NAD-linked); l lb , a-glycerolphosphate: Q6 oxidoreductase; 12, aspartate transaminase; 13, glutamate dehydrogenase; 14, dicarboxylate carrier; 15, phosphate cartier; 16, pyruvate transporter; 17, adenine nucleotide carrier; 18, ATP synthase; 19, alcoholdehydrogenase (ADHIII). NAD deh, internal and

external NADH : Q6 oxidoreductase, bc 1 : Q6H2: cytochrome c oxidoreductase; cco, cytochrome c oxidase.

232

growth on ethanol the enzymes induced include alcohol dehydrogenase (ADH II), aldehyde dehydrogenase, acetyl-CoA synthetase, the enzymes of the glyoxylate cycle, cytosolic malate dehydrogenase, phosphoenolpyruvate carboxykinase, en- olase (gluconeogenic form with a higher affinity for phosphoenolpyruvate) and fructose biphosphatase. The activity of the pyruvate carboxylase increases only by a factor of about 2 [168]. The induction of these enzymes is to some extent coregulated and occurs after release from glucose repression (cf. Refs. 164, 165 and 168). Induction of, for example, ADH II does not occur in petite mutants growing aerobically on glucose in the presence of ethanol or in mutants lacking the dicarboxylate carrier or the pyruvate transporter [244-246].

The quantitative aspects of growth on non-fermentable substrates and the mechanisms involved in the regulation of the cellular redox balance are not as well known as for growth on glucose. Fig. 7 shows an overview of intermediary metabolism of yeast growing on various non-fermentable carbon sources. The particular routes involved are as follows. Ethanol is first oxidized in two steps to acetate yielding NADH and NADPH, respectively. Acetate is converted to acetyl-CoA which is free to enter the Krebs cycle to meet the energy demands of the cell or the glyoxylate cycle. The net result of operation of the glyoxylate cycle is the formation of succinate and NADH. Succinate enters the mitochondrion in exchange for malate which in turn may be exchanged for phosphate and /o r O H - so as to replenish Krebs cycle intermediates. Gluconeogenesis starts with the conversion of malate via oxaloacetate into phosphoenolpyruvate catalysed by malate dehydrogenase and phosphoenolpyruvate carboxykinase [92-94]. Subsequently the pathway of glycolysis is reversed up to fructose-l,6-diphosphate. This compound is dephosphorylated by fructose biphosphatase yielding fructose-6-phosphate which in turn is isomerized to glucose-6-phosphate, the first intermediate of the pentose-phosphate pathway from which the biosynthesis of several cellular building blocks starts. For growth on lactate or pyruvate the oxaloacetate formed by the pyruvate carboxylase is used in gluconeogenesis or serves to replenish Krebs cycle intermediates. For growth

on glycerol, glycerol kinase and the mitochondrial (linked to Q) and the cytoplasmic (linked to NAD) a-glycerolphosphate dehydrogenases, normally repressed by glucose are induced allowing the formation of the triose phosphates. These latter are either converted to glucose-6-phosphate (fructose biphosphatase is induced) or metabolised via the normal glycolytic route. The conversion of pyruvate to oxaloacetate by pyruvate carboxylase is probably the main anaplerotic pathway for growth on glycerol.

In the analysis of the metabolic routes operat- ing during growth on non-fermentable carbon sources the activity of the malic enzyme was not considered. In fact, NADP-linked malate-de- carboxylating activity was reported to be absent in S. cerevisiae and C. utilis [22,168]. An NAD-linked malic-enzyme is present in Schizosaccharomyces pombe [209]. More recently, an NAD(P)-linked malic enzyme was partially purified from S. cerevisiae [74]. Its specific activity in the cell is 400 times lower than that of the cytosolic malate dehydrogenase and the K m values for malate and NAD are 50 mM and 0.5 mM, respectively. The physiological significance of the malic enzyme is therefore unclear. Furthermore, when present it would allow futile cycling between pyruvate, oxaloacetate and malate (cf. Ref. 92). Even the recent studies with a mutant having a tenfold reduced activity of the pyruvate carboxylase are not of much help in this matter (unfortunately, malic-enzyme activity was not determined) [246]. This mutant does not grow on minimal medium with ethanol or pyruvate, an observation which at first sight is consistent with the absence of the malic enzyme, since on ethanol there is no pathway to produce pyruvate, an important precursor in the synthesis of several amino acids, and on pyruvate no route to oxaloacetate exists. As expected then, the mutant did not grow on oxaloacetate whereas the wild type did. However, the mutant did not grow on a medium containing both ethanol and pyruvate, an observation that is difficult to understand. The mutant did grow on ethanol or pyruvate in the presence of aspartate. In the case of pyruvate, oxaloacetate is formed by t ransaminat ion of pyruvate and aspartate [92,93,246]. In the case of growth on ethanol it is still puzzling how pyruvate is formed. A possibil-

T A B L E VI

D I F F E R E N C E S B E T W E E N M I T O C H O N D R I A F R O M

Y E A S T A N D H I G H E R E U K A R Y O T E S

E n z y m e Y e a s t H i g h e r

e u k a r y o t e s

M i t C y t M i t C y t

T r a n s h y d r o g e n a s e - + ? + -

C o m p l e x I a _ _ + _

I n t e r n a l N A D H d e h y d r o g e n a s e

( n o s i te I) a + _ _ _

E x t e r n a l N A D H d e h y d r o g e n a s e a + _ _ _

F a t t y ac id o x i d a t i o n b _ + + +

I s o c i t r a t e d e h y d r o g e n a s e ( N A D P ) - + + +

M a l i c e n z y m e ( N A D ( P ) ) - - - +

G l u t a m a t e d e h y d r o g e n a s e ( N A D ( P ) ) - + + +

A s p a r t a t e a m i n o t r a n s a m i n a s e - + + +

M a l a t e / a s p a r t a t e s h u t t l e - +

P y r n v a t e c a r b o x y l a s e _ + c + _

L- lac ta te : c y t o c h r o m e c

o x i d o r e d u c t a s e + - - -

A l c o h o l d e h y d r o g e n a s e + + - +

A c e t a l d e h y d e d e h y d r o g e n a s e + + + -

c y t o c h r o m e c p e r o x i d a s e + - - -

a See a l so T a b l e I.

b C y t o s o l i c o x i d a t i o n o f f a t t y ac ids is r e s t r i c t e d to t h e pe r -

ox i somes .

c j . p . v a n D i j k e n ( p e r s o n a l c o m m u n i c a t i o n ) a n d Ref . 93.

ity is that now so much oxaloacetate is produced in the net oxidative decarboxylation of aspartate catalysed by the combined action of aspartate aminotransferase and glutamate dehydrogenase (see Fig. 7) that sufficient pyruvate is formed from phosphoenol p yruvate.

Table VI illustrates that mitochondria from yeast and higher eukaryotes differ in many re- spects from each other. These differences are general and not related to the species or organ referred to or the growth phase. The L-lactate : cytochrome c oxidoreductase, the cytochrome c peroxidase and the external and internal, nonphos- phorylating, NADH dehydrogenases are unique to yeast. Many of the differences listed are in one way or another related to the regulation of the mitochondrial and cytosolic redox balance as expressed in terms of the N A D / N A D H and N A D P / N A D P H redox couples. Therefore, their collective absence in yeast is possibly connected. For example the presence of an external NADH dehydrogenase in yeast mitochondria responsible

233

for the oxidation of cytosolic NADH eliminates the need for shuttle systems which in higher eukaryotes are involved in the transfer of excess reducing equivalents from the cytosol to the mitochondrion. The finding that the aspartate aminotransferase (and the glutamate dehydrogenase) is only present in the cytosol [104], thus precluding a functional malate-aspartate shuttle, is likely to be correlated with the presence of an external N A D H dehydrogenase. Similarly the fatty acid/malate-citrate shuttle (cf. Ref. 48) is presumably not important in yeast, the more so because mitochondrial r-oxidation of fatty acids is absent in almost all yeasts [64,117]. To what extent the a-glycerolphosphate shuttle, the a-oxo- glutarate/isocitrate shuttle, the malate shuttle and the ethanol/acetaldehyde shuttle are indeed important as shuttle systems in yeast in vivo remains to be established.

VII. Concluding remarks

In the last decade, the primary structures of many of the subunits constituting the respiratory- chain complexes have become available. From the primary structures, models for the secondary structures have been constructed. In spite of the fact that the predictive value of this procedure is generally rather low, it has contributed significantly to our understanding of some of the structural features that are important for function, especially in the case of membrane-bound polypeptides. It is clear, however, that knowledge of the three-dimensional structure of respiratory chain enzymes, obtained by X-ray crystallography, is a prerequisite to understand the structural requirements involved in the coupling of electron transfer to proton translocation. Once the three- dimensional structure is known, application of powerful techniques such as N M R and site-directed mutagenesis seems appropriate to obtain additional information on, e.g., the dynamics of the structure of these enzymes and to refine the various details of the structure/function relation.

The technique of random mutagenesis, either of total genomic DNA ' in vivo' or of a particular gene present on a plasmid ' in vitro', followed by screening for a particular phenotype, e.g., inhibitor resistance or thermosensitivity, and determina-

234

tion of the site of mutation, has so far yielded several interesting results concerning the function and stability of a polypeptide. However, owing to the lack of additional information on the three-dimensional structure it is not yet possible to draw conclusions on how and why these mutations cause their effects. The more novel approach of deletion or disruption of a gene, causing the complete absence of a subunit of a respiratory-chain enzyme, has provided information on the role of the subunits without prosthetic groups. The results obtained so far indicate that deletion of a single gene coding for a subunit of a respiratory-chain complex either has no (detectable) effect on assembly and activity, or results in a Complete loss of activity. The loss of activity is most likely due to the fact that the process of assembly is greatly disturbed, often resulting in a large decrease in the levels of the other subunits. Our proposal of the various stages of assembly of the bc 1 complex is necessarily speculative. Information on the sorting and location of several subunits is lacking, the mechanism of insertion of prosthetic groups is unknown and additional factors such as pantothenate in the case of assembly of cytochrome c oxidase (and of the ATP-synthase) may be required. Furthermore, whether subcomplexes are formed, as seems to be the case in the formation of, e.g., ribosomes, or not remains to be established. If they are, the question as to the composition of these subcomplexes has to be answered and, more generally, what are the driving forces causing some polypeptides to associate specifically.

It might have been anticipated from a comparison of the structural complexi ty of the respiratory-chain enzymes from prokaryotes with those from lower and higher eukaryotes that the additional subunits,, without prosthetic groups, present in the latter type of organisms do not have an essential role in electron transfer and proton translocation. This supposition has been borne out experimentally for a number of cases. Some of these subunits are apparently essential for assembly, but the 17 kDa protein of the b q complex and Cox8 of cytochrome c oxidase have neither a function in assembly nor in catalytic activity. A role for the subunits without prosthetic groups in the regulation of the activity has been proposed

(cf. Ref. 113). However, how this regulation is achieved and specifically why the process of oxidative phosphorylation should be regulated by factors in addition to the protonmotive force, the phosphate potential and the ATP-ase inhibitor in eukaryotes and not or to a far lesser extent in prokaryotes has not been worked out. Neverthe- less, the existence of isoenzymes of cytochrome c oxidase in higher eukaryotes, the presence of two types of subunit V of this enzyme in yeast and the finding that the concentration of cytosolic ATP modulates the activity of cytochrome c oxidase [106,182], indicate that this kind of regulation of the activity of the mitochondrial respiratory chain is somehow important in the control of cellular metabolism in eukaryotes. From this, one may conclude that mitochondria are not simply the energy plants of the cell. In fact, this is also inferred from the observation that mitochondria from, e.g., heart and fiver are markedly different. It is generally accepted that mitochondrial metabolism is also important in the generation of precursors of anabolic pathways. Furthermore, mitochondria may play a role in, e.g., Ca homeostasis of the cell, though not in the rapid release of Ca into the cytosol in response to certain hormones as is observed in higher eukaryotes (or possibly in response to certain 'external stimuli' in unicellular eukaryotes), but rather serve as a reservoir for Ca. In order to regulate and coordinate the rate of ATP synthesis, the rate of formation of precursors to anabolism and the rate of Ca efflux/influx, one can imagine that the respiratory-chain enzymes of prokaryotes have evolved to the much more complicated enzymes now found in eukaryotes, simply because the purely thermodynamic (chemiosmotic) control mechanism is not sufficiently sophisticated to cope with the function of mitochondria in the metabolism of a eukaryotic cell. Since cells in a multicellular organism com- municate with each other via, e.g., hormones, one can imagine that the respiratory-chain enzymes from the higher eukaryotes are even more complex that those from the lower eukaryotes.

We have listed the differences between mitochondria from yeast and from higher eukaryotes. Many of these differences are related to the regulation of the cellular redox balance and may therefore be evolutionarily correlated. In its

na tu ra l habi ta t , i.e., decaying fruit, yeas t (specif i- cal ly S. cerevisiae) metabol i ses ma in ly glucose, e thano l and lactate. In o rder to consume the la t te r two ca rbon sources, yeas t has acqui red specific enzymes and has, in con t ras t to the h igher eukaryotes , r e ta ined the g lyoxyla te cycle. F ree fa t ty acids will be rare in decaying fruit and it is

therefore no t surpr is ing that the p a t h w a y of mi tochondr i a l fa t ty acid ox ida t ion is absen t in

mos t yeasts. Some of the metabo l i c steps that take p lace in the m i t o c h o n d r i a of higher eukaryotes , occur in yeas t in the cytoplasm. In addi t ion , yeas t conta ins cy tochrome c peroxidase , bu t appa ren t l y lacks a mi tochondr i a l t ranshydrogenase . Some of these differences are no t easi ly unders tood , b u t cer ta in ly make clear tha t e luc ida t ion of the various aspects of mi tochondr i a l me t abo l i sm will form an inspi r ing area for fu ture research.

Acknowledgements

W e thank Dr. S.P.J. Albrach t , J.A. Berden, L.A. Grivell , A.J. Meyer , A.O. Muysers and P.J. Schopp ink for read ing the manuscr ip t . W e also thank Drs. A. -M. Colson, J.P. di Rago, W. Hemrika , P.O. Ljungdahl , J. Philips, P.J. Schop- p ink and B.L. T r u m p o w e r for mak ing avai lab le to us their exper imenta l results and pape r s p r io r to publ ica t ion . Par t of this work was suppor t ed b y a g ran t (to S. de V.) f rom the Roya l D u t c h A c a d e m y

of Sciences (K.N.A.W.) .

References

1 Albracht, S.P.J. and Bakker, P.T.A. (1986) Biochim. Bio- phys. Acta 850, 423-428.

2 Albracht, S.P.J. and Subramanian, J. (1977) Biochim. Bio- phys. Acta 462, 36-48.

3 Alliel, P.M., Guiard, B., Ghrir, R., Becam, A.-M. and Lederer, F. (1982) Eur. J. Biochem. 122, 553-558.

4 Anderson, S., Bankier, A.T., Barrell, B.G., De Bruijn, M.H.L., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H., Staden, R. and Young, I.G. (1981) Nature, 290, 457-465.

5 Anderson, S., De Bruijn, M.H.L., Coulson, A.R., Eperon, I.C., Sanger, F. and Young, I:G. (1982) J. Mol. Biol. 156, 683-717.

6 Autor, A.P. (1982) J. Biol. Chem. 257, 2713-2718. 7 Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Bio-

phys. Acta 850, 413-422. 8 Beckmann, J.D., Ljungdahl, P.O., Lopez, J.L. and Trum-

power, B.L. (1987) J. Biol. Chem. 262, 8901-8909.

235

9 Beinert, H. and Albracht, S.P.J. (1982) Biochim. Biophys. Acta 683, 245-277.

10 Berden, J.A., Van Keulen, M. and Marres, C.A.M. (1985) in Proton Pumping in Respiration and Photosynthesis, Table Ronde Roussel-Uclaf, no. 52, Paris, pp. 51-52.

11 Blair, D.F., Martin, C.T., Gelles, J., Wang, H., Brudvig, G.W., Stevens, T.H. and Chan, S.I. (1983) Chem. Script. Vol. 21, 43-53.

12 Boelens, R., Wever, R., Van Gelder, B.F. and Rademaker, H. (1983) Biochim. Biophys. Acta 724, 176-183.

13 Bonitz, S.G., Coruzzi, C., Thalenfeld, B.E., Tzagoloff, A. and Macino, G. (1980) J. Biol. Chem. 255, 11927-11941.

14 Borchart, U., Machleidt, W., Sch~igger, H., Link, T.A. and Von Jagow, G. (1986) FEBS Lett. 191, 125-]29.

15 Borchart, U., Machleidt, W., Sch~igger, H., Link, T.A. and Von Jagow, G. (1986) FEBS Lett. 200, 81-86.

16 Brambl, R. and Plesofski-Vig, N. (1986) Proc. Natl. Acad. Sci. USA 83, 3644-3648.

17 Breitenberger, C.A. and RajBhandary, U.L. (1985) TIBS 10, 478-482.

18 Briquet, M., Purnelle, B., Beattie, D.S. and Goffau, A. (1982) Enr. J. Biochem. 127, 339-342.

19 Broger, C., Salardi, S. and Azzi, A. (1983) Eur. J. Bio- chem. 131, 349-352.

20 Bruinenberg, P.M., Van Dijken, J.P., Kuenen, J.G. and Scheffers, W.A. (1985) J. Gen. Microbiol. 131, 1043-1051.

21 Bruinenberg, P.M., Van Dijken, J.P. and Scheffers, W.A. (1983) J. Gen. Microbiol. 129, 953-964.

22 Bruinenberg, P.M., Van Dijken, J.P. and Scheffers, W.A. (1983) J. Gen. Microbiol. 129, 965-971.

23 Bruinenberg, P.M., Jonker, R., Van Dijken, J.P. and Scheffers, W.A. (1985) Arch. Microbiol. 142, 302-306.

24 Brunori, M., Antonini, E. and Wilson, M.T. (1981) in Metal Ions in Biological Systems (Sigel, M., ed.), pp. 115-142, Dekker, New York.

25 Capaldi, R.A., Gonzalez-Halphen, D. and Takamiya, S. (1986) FEBS Lett. 207, 11-17.

26 Capaldi, R.A., Malatesta, F. and Darley-Usmar, V.M. (1983) Biochim. Biophys. Acta 726, 135-148.

27 Capaldi, R.A., Takamyia, S., Zhang, Y.-Z., Gonzalez- Halphen, D. and Yanamura, W. (1987) Curr. Top. Bioen- erg. 15, 91-113.

28 Capeill~re-Blandin, C. (1975) Eur. J. Biochem. 56, 91-101. 29 Capeill6re-Blandin, C. (1982) Eur. J. Biochem. 128,

533-542. 30 Capeill~re-Blandin, C., Bray, R., Iwatsubo, M. and

Labeyrie, F. (1975) Eur. J. Biochem. 54, 549-566. 31 Capeillare-Blandin, C. and Ohnishi, T. (1982) Eur. J.

Biochem. 122, 403-413. 32 Casey, R.P., Thelen, M. and Azzi, A. (1980) J. Biol. Chem.

255, 3994-4000. 33 Charalampous, F.C. and Chen, W.L. (1974) J. Biol. Chem.

249, 1007-1013. 34 Chen, S. and Guillory, R.J. (1981) J. Biol. Chem. 256,

8318-8332. 35 Chevillotte-Brivet, P., Meunier-Lemesle, D., Forget, N.

and Pajot, P. (1983) Eur. J. Biochem. 129, 653-661.

236

36 Chomyn, A. and Attardi, G. (1987) Curt. Top. Bioenerg. 15, 295-331.

37 Chomyn, A., Mariottini, P., Cleeter, M.W.J., Ragan, C.I., Matsuno-Yagi, A., Hatefi, Y., Doolittle, R.F. and Attardi, G. (1985) Nature 314, 592-597.

38 Cline, J.F., Hoffman, B.M., Mires, W.B., LaHaie, E., Ballou, D.P. and Fee, J.A. (1985) J. Biol. Chem. 260, 3251-3254.

39 Cobley, J.G., Grossman, S. and Singer, T.P. (1975) J. Biol. Chem. 250, 211-217.

40 Coruzzi, G. and Tzagoloff, A. (1979) J. Biol. Chem. 254, 9324-9330.

41 Crofts, A.R. (1986) J. Bioenerg. Biomembr. 18, 437-445. 42 Crofts, A.R., Robinson, H. and Andrews, K. (1987) in

Cytochrome Systems (Papa, S., Chance, B. and Ernster, L., eds.), pp. 617-624, Plenum Press, New York and London.

43 Cumski, M.G., Trueblood, C.E., Ko, C. and Poyton, R.O. (1987) Mol. Cell. Biol. 7, 3511-3519.

44 Daldal, F., Davidson, E. and Cheng, S. (1987) J. Mol. Biol. 195, 1-12.

45 Daum, G., B/Shni, P.C. and Schatz, G. (1982) J. Biol. Chem. 257, 13028-13033.

46 Davidson, E. and Daldal, F. (1987) J. Mol. Biol. 195, 13-24.

47 Davis, K.A., Mackler, B., Haynes, B., Person, R., Bevan, C., Grace, R., Palmer, G. and Carter, K. (1982) Biochem. Int. 5, 49-54.

48 Dawson, A.G. (1979) TIBS 4, 171-176. 49 Degli Esposti, M., Ballester, F., Solaini, G. and Lenaz, G.

(1987) Biochem. J. 241, 285-290. 50 De Haan, M., Van Loon, A.P.G.M., Kreike J., Vaessen,

R.T.M.J. and Grivell, L.A. (1984) Eur. J. Biochem. 138, 169-177.

51 De Santis, A. and Melandri, B.A. (1984) Arch. Biochem. Biophys. 232, 354-365.

52 De Vries, H., Alzner-De Weerd, B., Breitenberger, C.A., Chang, D.D., De Jonge, J.C. and RajBhandary, U.L. (1986) EMBO J. 5, 779-785.

53 De Vries, S. (1986) J. Bioenerg. Biomembr. 18, 195-224. 54 De Vries, S., Albracht, S.P.J., Berden, J.A., Marres, C.A.M.

and Slater, E.C. (1983) Biochim. Biophys. Acta 723, 91-103.

55 De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 41-53.

56 De Vries, S., van Hoek, A.N. and Berden, J.A. (1988) Biochim. Biophys. Acta 935, 208-216.

57 De Vries, S. and Grivell, L.A. (1982) in Membrane Bio- chemistry and Bioenergetics (Kim, C.H., Tedeschi, H., Diwan, J.J. and Salerno, J.C., eds.), pp. 115-119, Plenum Press, New York and London.

58 De Vries, S. and Grivell, L.A. (1988) Eur. J. Biochem. 176, 377-384.

59 De Zamarocczy, M. and Bernardi, G. (1985) Gene 37, 1-17.

60 Di Rago, J.P. and Colson, A.-M. (1988) J. Biol. Chem. 263, 12564-12570.

61 Di Rago, J.P., Perea, X. and Colson, A.-M. (1986) FEBS Lett. 208, 208-210.

62 Djavadi, F.H.S., Moradi, M. and Djavadi-Ohaniance, L. (1980) Eur. J. Biochem. 107, 501-504.

63 Dobberstein, B., Blobel, G. and Chua, N.-H. (1977) Proc. Natl. Acad. Sci. USA 74, 1082-1085.

64 Dommes, P., Dommes, V. and Kunau, W.-H. (1983) J. Biol. Chem. 258, 10846-10852.

65 Dooijewaard, G., De Bruin, G.J.M., Van Dijk, P.J. and Slater, E.C. (1978) Biochim. Biophys. Acta 501, 458-469.

66 Dowhan, W., Bibus, C.R. and Schatz, G. (1985) EMBO J. 4, 179-184.

67 Evans, T.C., Mackler, B. and Grace, R. (1985) Arch. Biochem. Biophys. 243, 492-503.

68 Fee, J.A., Findling, K.L., Yoshida, T., Hille, R., Tan', E., Hearshen, D.O., Dunham, W.R., Day, E.P., Kent, A. and Miinck, E. (1984) J. Biol. Chem. 259, 124-133.

69 Fee, J.A., Kuila, D., Mather, M.W. and Yoshida, T. (1986) Biochim. Biophys. Acta 853, 153-185.

70 Ferrero, I., Saccani, M.G., Rossi, C., Viola, A.M. and Puglisi, P.P. (1981) Microbiologica 4, 131-139.

71 Fox, T.D. (1979) Proc. Natl. Acad. Sci. USA 76, 6534-6538.

72 Fraenkel, D.G. (1982) in The Molecular Biology of Yeast (Strathern, J.N., Jones, E.W., Broach, J.R., eds.), pp. 1-37, Cold Spring Harbour Laboratory, New York.

73 Fraenkel, D.G. (1986) Ann. Rev. Biochem. 55, 317-337. 74 Fuck, E., St~irk, G. and Radler, F. (1973) Arch. Mikrobiol.

89, 223-231. 75 Gabellini, N. and Sebald, W. (1986) Eur. J. Biochem. 154,

569-579. 76 Galante, Y.M. and Hatefi, Y. (1979) Arch. Biochem.

Biophys. 192, 559-568. 77 Gasser, S.M., Ohashi, A., Danm, G., B/Shni, P.C., Gibson,

J., Reid, G.A., Yonetani, T. and Schatz, G. (1982) Proc. Natl. Acad. Sci. USA 79, 267-271.

78 Gbelska, Y., Subik, J., Svoboda, A., Goffau, A. and Kovac, L. (1983) Eur. J. Biochem. 130, 281-286.

79 Genga, A.M., Tassi, F., Lodi, T. and Ferrero, I. (1983) Microbiologica 1, 1-8.

80 Gervais, M., Labeyrie, F., Risler, Y. and Vergnes, O. (1980) Eur. J. Biochem. 111, 17-31.

81 Gervais, M., Risler, Y. and Corazzin, S. (1983) Eur. J. Biochem. 130, 253-259.

82 Gervais, M. and Tegoni, M. (1980) Eur. J. Biochem. 111, 357-367.

83 Gollub, E.C. and Dayan, J. (1985) Biochem. Biophys. Res. Commun. 128, 1447-1454.

84 Gray, J.C. (1978) Eur. J. Biochem. 82, 133-141. 85 Gregolin, C. and Singer, T. (1962) Biochim. Biophys. Acta

67, 201-218. 86 Grivell, L.A. (1983) in Mitochondria. Nucleo-

Mitochondrial Interactions (Schweyen, R.J., Wolf, K. and Kaudewitz, F., eds.), pp. 25-45, De Gruyter, Berlin.

87 Grivell, L.A. (1988) Int. Rev. Cytol. 3, 107-139. 88 Grossman, S., Cobley, J.G. and Singer, T.P. (1974) J. Biol.

Chem. 249, 3819-3826. 89 Guiard, B. (1985) EMBO J. 4, 3265-3272. 90 Guiard, B., Groudinsld, O. and Lederer, F. (1974) Proc.

Natl. Acad. Sci. USA 71, 2539-2543. 91 Guiard, B. and Lederer, F. (1975) Nature 255, 422-423.

92 Haarasilta, S. and Oura, E. (1975) Eur. J. Biochem. 52, 1-7.

93 Haarasilta, S. and Taskinen, L. (1977) Arch. Microbiol. 113, 159-161.

94 H~igele, E., Neeff, J. and Mecke, D. (1978) Eur. J. Bio- chem. 83, 67-76.

95 Hallermayer, G., Zimmerman, R. and Neupert, W. (1977) Eur. J. Biochem. 81, 523-532.

96 Harmey, M.A. and Neupert, W. (1985) in The Enzymes of Biological Membranes (Martonosi, A., ed.), Vol. 4, pp. 431-464, Plenum Press, New York.

97 Harnisch, U., Weiss, H. and Sebald, W. (1985) Eur. J. Biochem. 149, 95-99.

98 Hartl, F.-U., Schmidt, B. and Neupert, W. (1986) Biol. Chem. Hoppe Seyler, 367, 173.

99 Hartl, F.-U., Ostermann, J., Guiard, B. and Neupert, W. (1987) Cell 51, 1027-1037.

100 Hartl, F.-U., Schmidt, B., Wachter, E., Weiss, H. and Neupert, W. (1986) Cell 47, 939-951.

101 Hauska, G., Hurt, E., Gabellini, N. and Lockau, W. (1983) Biochim. Biophys. Acta 726, 93-133.

102 Hay, R., BiShni, P. and Gasser, S. (1984) Biochim. Bio- phys. Acta 779, 65-87.

103 Heron, C., Smith, S. and Ragan, C.I. (1979) Biochem. J. 181, 435-443.

104 Hollenberg, C.P., Riks, W.F. and Borst, P. (1970) Bio- chim. Biophys. Acta 201, 13-19.

105 H01m, L., Saraste, M. and Wikstr~Sm, M. (1987) EMBO J. 6, 2819-2823.

106 Hilther, F.-J. and Kadenbach, B. (1986) FEBS Lett. 207, 89-94.

107 Ise, W., Haiker, H. and Weiss, H. (1985) EMBO J. 4, 2075-2080.

108 Iwatsubo, M., M6vel-Ninio, M. and Labeyrie, F. (1977) Biochem. 16, 3558-3566.

109 Jacq, C. and Lederer, F. (1974) Eur. J. Biochem. 41, 311-320.

110 Japa, S, Zhu, Q.-S. and Beattie, D.S. (1987) J. Biol. Chem. 262, 5441-5444.

111 Johanningmeier, U., Bodner, U. and Wildner, G.F. (1987) FEBS LetL 211,221-224.

112 Joliot, P. and Joliot, A. (1986) Biochim. Biophys. Acta 849, 211-222.

113 Kadenbach, B., Kuhn-Nentwig, L. and Bilge, U. (1987) Curr. Top. Bioenerg. 15, 114-162.

114 Kadenbach, B., Ungibauer, M., Jarausch, J. Bilge, U. and Kuhn-Nentwig, LI (1983) TIBS 8, 398-400.

115 Karlsson, B., HovrnNler, S., Weiss, H. and Leonard, K. (1983) J. Mol. Biol. 165, 287-302.

116 Katan, M.B., Pool, L. and Groot, G.S. (1976) Eur. J. Biochem. 65, 95-105.

117 Kawamoto, S., Nozaki, C., Tanaka, A. and Fukui, S. (1978) Eur. J. Biochem. 83, 609-613.

118 Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108.

119 King, T:E: and Kim, C.H. (1983) J. Biol. Chem. 258, 13543-13551.

120 Koerner, T.J., Hill, J. and Tzagoloff, A. (1985) J. Biol. Chem. 260, 9513-9515.

237

121 Koerner, T.J., Homison, G. and Tzagoloff, A. (1985) J. Biol. Chem. 260, 5871-5874.

122 Kreike, J., Bechmann, H., Van Hemert, F.J., Schweyen, R.J., Boer, P.H., Kaudewitz, F. and Groot, G.S.P. (1979) Eur. J. Biochem. 101, 607-617.

123 Kurowski, B. and Ludwig, B. (1987) J. Biol. Chem. 262, 13805-13811.

124 Labeyrie, F., Bandras, A. and Lederer, F. (1978) Methods Enzymol. 53, 238-256.

125 Lagunas, R. (1976) Biochim. Biophys. Acta 440, 661-674. 126 Lagunas, R. (1979) Mol. Cell. Biochem. 27, 139-146. 127 Lagunas, R. (1981) TIBS 6, 201-203. 128 Lagunas, R. and Gancedo, J.M. (1973) Eur. J. Biochem.

37, 90-94. 129 Leonard, K., Haiker, H. and Weiss, H. (1987) J. Mol. Biol.

194, 277-286. 130 Li, Y., Leonard, K. and Weiss, H. (1981) Eur. J. Biochem.

116, 199-205. 131 Li, Y., de Vries, S., Leonard, K. and Weiss, H. (1981)

FEBS Lett. 135, 277-280. 132 Ljungdahl, P.O. (1987) in Mutational Analysis of the

Mitochondrial Rieske Iron-Sulphur Protein of Sac- charomyces cerevisiae. PhD Thesis, Dartmouth College, Hanover, NH.

133 Ljungdahl, P.O., Pennoyer, J.D., Robertson, D.E. and Trumpower, B.L. (1987) Biochim. Biophys. Acta 891, 227-241.

134 Ludwig, B. (1987) FEMS Microbiol. Rev. 46, 41-56. 135 Maarse, A.C. and Grivell, L.A. (1987) Eur. J. Biochem.

165, 419-425. 136 Maarse, A.C., De Haan, M., Schoppink, P.J., Berden, J.A.

and Grivell, L.A. (1988) Eur. J. Biochem. 172, 17%184. 137 Maarse, A.C., Van Loon, A.P.G.M., Riezman, H., Gregor,

I., Schatz, G. and Grivell, L.A. (1984) EMBO J. 3, 2831-2837.

138 Maccecchini, M.-L., Rudin, Y., Blobel, G. and Schatz, G. (1979) Proc. Natl. Acad. Sci. USA 76, 343-347.

139 Mackler, B., Bevan, C., Person, R. and Davis, K.A. (1981) Biochem. Int. 3, 9-17.

140 Mackler, B. and Haines, B. (1973) Biochim. Biophys. Acta 292, 88-91.

141 Mackler, B., Haines, B., Person, R. and Palmer, G. (1980) Biochim. Biophys. Acta 591,289-297.

142 Marres, C.A.M. (1983) in Subunit structure and function of ubiquinol: cytochrome c oxidoreductase, Ph.D. Thesis, University of Amsterdam, Rodopi, Amsterdam.

143 Marres, C.A.M., De Vries, S. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 323-326.

144 Marres, C.A.M. and Slater, E.C. (1977) Biochim. Biophys. Acta 462, 531-548.

145 Marres, C.A.M., van Loon, A.P.G.M., Oudshoorn, P., Van Steeg, H., Grivell, L.A. and Slater, E.C. (1985) Eur. J. Biochem. 147, 153-161.

146 McEwen, .I.E., Ko, C., Kloeckner-Gruissem, B. and Poy- ton, R.O. (1986) J. Biol. Chem. 261, 11872-11879.

147 Meier, E.M., Schuitenmaker, M.G., Boogerd, F.C., Wever, R. and Stouthamer, A.H. (1978) Arch. Microbiol. 119, 119-127.

238

148 Meunier-Lemesle, D., Chevilotte-Brivet, P. and Pajot, P. (1980) Eur. J. Biochem. 111, 151-159.

149 Michel, H., Epp, O. and Deisenhofer, J. (1986) EMBO J. 5, 2445-2451.

150 Mihara, K. and Blobel, G. (1980) Proc. Natl. Acad. Sci. USA 77, 4160-4164.

151 Mitchell, P. (1976) J. Theor. Biol. 62, 327-367. 152 Moss, D.A. and Rich, P.R. (1987) Biochim. Biophys. Acta

894, 189-197. 153 Myers, A.M., Crivellone, M.D., Koerner, T.J. and Tzago-

loft, A. (1987) J. Biol. Chem. 262, 16822-16829. 154 Nelson, N. and Schatz, G. (1979) Proc. Natl. Acad. Sci.

USA 76, 4365-4369. 155 Neupert, W. and Von R~cker, A. (1976) In Genetics and

Biogenesis of Chloroplasts and Mitochondria (Biicher, T., Neupert, W., Sebald, W. and Werner, S., eds.), pp. 231-238, Elsevier/North-Holland, Amsterdam.

156 Nobrega, F.G. and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837.

157 Ohnishi, T. (1970) Biochem. Biophys. Res. Commun. 41, 344-352.

158 Ohnishi, T. (1973) Biochim. Biophys. Acta 301, 105-128. 159 Ohnishi, T., Asakura, T., Wohlrab, H., Yonetani, T. and

Chance, B. (1970) J. Biol. Chem. 245, 901-902. 160 Ohnishi, T. and Trumpower, B.L. (1980) J. Biol. Chem.

255, 3278-3284. 161 Oudshoorn, P., Van Steeg, H., Swinkels, B.W., Schoppink,

P.J. and Grivell, L.A. (1987) Eur. J. Biochem. 163, 97-103. 162 Pajot, P. and Claisse, M.L. (1974) Eur. J. Biochem. 49,

275-285. 163 Patterson, T.E. and Poyton, R.O. (1986) J. Biol. Chem.

261, 17192-17197. 164 Perlman, P. and Mahler, H.R. (1970) Arch. Biochem.

Biophys. 136, 245-259. 165 Perlman, P. and Mahler, H.R. (1974) Arch. Biochem.

Biophys. 162, 248-271. 166 Pfanner, N. and Neupert, W. (1987) Curt. Top. Bioenerg.

15, 178-221. 167 Pfefferkorn, B. and Meyer, H.E. (1986) Eur. J. Biochem.

206, 233-237. 168 Polakis, E.S. and Bartley, W. (1965) Biochem. J. 97,

284-302. 169 Pompon, D. (1980) Eur. J. Biochem. 106, 151-159. 170 Pompon, D., Iwatsubo, M. and Lederer, F. (1980) Eur. J.

Biochem. 104, 479-488. 171 Power, S.D., Lochrie, M.A. Patterson, T.E. and Poyton,

R.O. (1984) J. Biol. Chem. 259, 6571-6574. 172 Power, S.D., Lochrie, M.A. and Poyton, R.O. (1984) J.

Biol. Chem. 259, 6575-6578. 173 Power, S.D., Lochrie, M.A. and Poyton, R.O. (1986) J.

Biol. Chem. 261, 9206-9209. 174 Power, S.D., Lochrie, M.A., Sevarino, K.A., Patterson,

T.E. and Poyton, R.O. (1984) J. Biol. Chem. 259, 6564-6570.

175 Powers, L. (1982) Biochim. Biophys. Acta 683, 1-38. 176 Powers, L., Chance, B., Ching, Y. and Angilillo, P. (1981)

Biophys. J. 34, 465-498. 177 Poyton, R. and Schatz, G. (1975) J. Biol. Chem. 250,

752-761.

178 Ragan, C.I. (1987) Curr. Top. Bioenerg. 15, 1-36. 179 Raito, M., Jalli, T. and Saraste, M. (1987) EMBO J. 6,

2825-2833. 180 Rich, P.R. (1984) Biochim. Biophys. Acta 768, 53-79. 181 Rich, P.R. and Wikstr~Sm, M. (1986) FEBS Lett. 194,

176-182. 182 Rigoulet, M. , Guerin, B. and Denis, M. (1987) Eur. J.

Biochem. 168, 275-279. 183 RiSmisch, J., Tropschug, M., Sebald, W. and Weiss, H.

(1987) Eur. J. Biochem. 164, 111-115. 184 Rubin, M.S. and Tzagoloff, A. (1973) J. Biol. Chem. 248,

4269-4274. 185 Sadler, I., Suda, K., Schatz, G., Kaudewitz, F. and Haid,

A. (1984) EMBO J. 3, 2137-2143. 186 Saraste, M. 0984) FEBS Lett. 166, 367-372. 187 SchSgger, H., Von Jagow, G., Borchart, U. and Machleidt,

W. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 307-311. 188 Sch~igger, H., Borchart, U., Aquila, H., Link, Th.A. and

Von Jagow, G. (1985) FEBS Lett. 190, 89-94. 189 Sch~igger, H., Borchart, U., Machleidt, W., Link, T.A. and

Von Jagow, G. (1987) FEBS Lett. 219, 161-168. 190 Schatz, G. (1979) FEBS Lett. 103, 203-211. 191 Sehatz, G. (1981) in Mitochondria and Microsomes (Lee,

C.P., Schatz, G. and Dallner, G., eds.), pp. 45-66, Addison-Wesley, Reading, MA.

192 Schleyer, M. and Neupert, W. (1985) Cell 43, 339-350. 193 Schleyer, M., Schmidt, B. and Neupert, W. (1982) Eur. J.

Biochem. 125, 109-116. 194 Schoppink, P.J., Grivell, L.A. and Berden, J.A. (1987) in

Membrane Biochemistry and Bioenergetics (Kim, C.H., Tedeschi, H., Diwan, J.J. and Salerno, J.C., eds.), pp. 129-140, Plenum Press, New York and London.

195 Schoppink, P.J., Hemrika, W., Reynen, J.M., Grivell, L.A. and Berden, J.A. (1988) Eur. J. Biochem. 173, 115-122.

196 Schwitzgu~bel, N.-P. and Palmer, J.M. (1982) J. Bacteriol. 149, 612-619.

197 Siedow, J.N., Power, S., De la Rosa, F.F. and Palmer, G. (1978) J. Biol. Chem. 253, 2392-2399.

198 Siedow, J.N., Vickery, L.E. and Palmer, G. (1980) Arch. Biochem. Biophys. 203, 101-107.

199 Slater, E.C. (1981) in Chemiosmotic Proton Circuits in Biological Membranes (Skulachev, V.P. and Hinkle, P.C., eds.), pp. 69-104, Addison-Wesley, Reading, MA.

200 Steinriicke, P., Steffens, G.C.M., Panskus, G., Buse, G. and Ludwig, B. (1987) Eur. J. Biochem. 167, 431-439.

201 Stepputm, J., Rother, C., Hermans, J., Jansen, T., Salni- kow, J., Hauska, G. and Herrmann, R.G. (1987) Mol. Gen. Genet. 210, 171-177.

202 Stonehuerner, J , O'Brien, P., Geren, L., Milett, F., Steidl, J., Yu, L. and Yu, C.-A. (1985) J. Biol. Chem. 260, 5392-5398.

203 Takamyia, S., Lindorfer, M.A. and Capaldi, R.A. (1987) FEBS Lett. 218, 277-282.

204 Tegoni, M., Janot, J.M. and Labeyrie, F. (1986) Eur. J. Biochem. 155, 491-503.

205 Tegoni, M., Janot, J.M., Silvestrini, M.C., Brunori, M. and Labeyrie, F. (1984) Biochem. Biophys. Res. Commun. 118, 753-759.

206 Tegoni, M., Silvestrini, M.C., Labeyrie, F. and Brunori, M. (1984) Eur. J. Biochem. 140, 39-45.

207 Teintze, M., Slaughter, M., Weiss, H. and Neupert, W. (1982) J. Biol. Chem. 257, 10364-10371.

208 Telser, J., Hoffman, B.M., LoBrutto, R., Ohnishi, T., Tsai, A.-L., Simpkin, D. and Palmer, G. (1987) FEBS Lett. 214, 117-121.

209 Temperli, A., Kiinsch, U., Mayer, K., Busch, L. (1965) Biochim. Biophys. Acta 110, 630-632.

210 Tempest, D.W. and Neyssel, O.M. (1984) Annu. Rev. Microbiol. 38, 459-486.

211 Thalenfeld, B.E. and Tzagoloff, A. (1980) J. Biol. Chem. 255, 6163-6180.

212 Thierbach, G. and Michaelis, G. (1982) Mol. Gen. Genet. 186, 501-506.

213 Thomas, M.-A., Favaudon, V. and Pochon, F. (1983) Eur. J. Biochem. 135, 569-576.

214 Thomas, M.-A., Gervais, M., Favaudon, V. and Valat, P. (1983) Eur. J. Biochem. 135, 577-581.

215 Trueblood, C.E and Poyton, R.O. (1987) Mol. Cell. Biol. 7, 3520-3526.

216 Tzagoloff, A., Akai, A. and Needleman, R.B. (1975) J. Biol. Chem. 250, 8228-8235.

217 Tzagoloff, A., Wu, M. and Crivellone, M. (1986) J. Biol. Chem. 261, 17163-17169.

218 Urban, Ph., Alliel, P.M. and Lederer, F. (1983) Eur. J. Biochem. 134, 275-281.

219 Urban, Ph. and Lederer, F. (1984) Eur. J. Biochem. 144, 345-351.

220 Urban, Ph. and Lederer, F. (1985) J. Biol. Chem. 260, 11115-11122.

221 Van Dijken, J.P. and Scheffers, W.A. (1986) FEMS Micro- biol. Rev. 32, 199-224.

222 Van Loon, A.P.G.M., Br~ndli, A.W., Pesold-Hurt, B., Blank, D. and Schatz, G. (1987) EMBO J. 6, 2433-2439.

223 Van Loon, A.P.G.M., De Groot, R.J., De Haan, M., Dekker, A. and GriveU, L.A. (1984) EMBO J. 3, 1039-1043.

224 Van Loon, A.P.G.M., De Groot, R.J., Van Eijk, E., Van der Horst, G.T.J. and Grivell, L.A. (1982) Gene 20, 323-327.

225 Van Loon, A.P.G.M., Kreike, J., De Ronde, A., Van der Horst, G.T.J., Gasser, S.M. and Grivell, L.A. (1983) Eur. J. Biochem. 135, 457-463.

226 Van Loon, A.P.G.M., Maarse, A.C., Riezman, H. and Grivell, L.A. (1983) Gene 26, 261-272.

227 Van Loon, A.P.G.M. and Schatz, G. (1987) EMBO J. 6, 2441-2448.

228 Viard, I. and Stahl, A.J.C. (1985) FEBS Lett. 180, 117-121. 229 Von Jagow, G. and Engel, W.D. (1981) FEBS Lett. 136,

19-24. 230 Von Jagow, G. and Klingenberg, M. (1970) Eur. J. Bio-

chem. 12, 538-592.

239

231 Von Jagow, G., Link, T.A., Ohnishi, T. and Sch~igger, H. (1985) in Achievements and Perspectives of Mitochondrial Research (Quagliariello, E., Slater, E.C., Palmieri, F., Sac- cone, C. and Kroon, A.M., eds.), Vol. I, pp. 115-126, Elsevier, Amsterdam.

232 Wakabayashi, S., Matsubara, H., Kim, C.H., Kawai, K. and King, T.E. (1980) Biochem. Biophys. Res. Commun. 97, 1548-1554.

233 Wakabayashi, S., Takao, T., Shimonishi, Y., Kuramitsu, S., Matsubara, H., Wang, T., Zang, Z. and King, T.E. (1985) J. Biol. Chem. 260, 337-343.

234 Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C.H. and King, T.E. (1982) J. Biochem. 91, 2077-2085.

235 Weiss, H. (1987) Curt. Top. Bioenerg. 15, 67-90. 236 Weiss, H. and Kolb, H.J. (1979) Eur. J. Biochem. 99,

139-149. 237 Widger, W.R., Cramer, W.A., Herrmann, R.G. and Trebst,

A. (1984) Proc. Natl. Acad. Sci. USA 81, 674-678. 238 Wikstr~Sm, M. and Krab, K. (1979) Biochim. Biophys.

Acta 549, 177-222. 239 Wikstr~Sm, M. and Krab, K. (1986) J. Bioenerg. Bio-

membr. 18, 181-193. 240 Wikstr~Sm, M., Saraste, M. and Krab, K. (eds.) (1981)

Cytochrome Oxidase: A Synthesis, Academic Press, New York.

241 Wikstr~Sm, M., Saraste, M. and Penttil~i, T. (1985) in The Enzymes of Biological Membranes (Martonosi, A.N., ed.), Vol. 4, pp. 111-148, Plenum, New York.

242 Willey, D.L., Howe, C.J., Auffret, A.D., Bowman, C.M., Dyer, T.A. and Gray, J.C. (1984) Mol. Gen. Genet. 194, 416-422.

243 Willey, D.L., Auffret, A.D. and Gray, J.C. (1984) Cell 36, 555-562.

244 Wills, C. and Martin, T. (1984) Biochim. Biophys. Acta 782, 274-284.

245 Wills, C., Martin, T. and Melham, T. (1986) Arch. Bio- chem. Biophys. 246, 306-320.

246 Wills, C. and Melham, T. (1985) Arch. Biochem. Biophys. 236, 782-791.

247 Wright, R.M., Ko, C., Cumski, M.G. and Poyton, R.O. (1984) J. Biol. Chem. 259, 15401-15407.

248 Wright, R.M., Dircks, L.K. and Poyton, R.O. (1986) J. Biol. Chem. 261, 17183-17191.

249 Yagi, T., Hon-ami, K. and Ohnishi, T. (1988) Biochem- istry 27, 2008-2013.

250 Yang, X. and Trumpower, B.L. (1986) J. Biol. Chem. 261, 12282-12289.

251 Zhu, Q.-S. and Beattie, D.S. (1988) J. Biol. Chem. 263, 193-199.

252 Zimmerman, R. and Meyer, D.I. (1986) TIBS 11, 512-515.

the mitochondrial respiratory chain of yeast. structure and biosynthesis and the role in cellular...

Documents