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MICROBIOLOGICAL REVIEWS, June 1985, p. 140-157 Vol. 49, No. 20146-0749/85/020140-18$02.00/0Copyright © 1985, American Society for Microbiology

In Bacteria Which Grow on Simple Reductants, Generation of aProton Gradient Involves Extracytoplasmic Oxidation of Substrate

ALAN B. HOOPER* AND ALAN A. DISPIRITOt

Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108

INTRODUCTION..................................................... 140Topology..................................................... 141Strategies for Establishment of a Proton Gradient ..................... ................................ 141

Direct "translocation" of H+..................................................... 141Translocation of H through "redox loops"...................................................... 141Cytoplasmic H+-utilizing electron acceptors ............... ...................................... 141Extracytoplasmic oxidation of simple substrates ..................... ................................ 142

EXAMPLES OF EXTRACELLULAR OXIDATION OF SUBSTRATE ........................................... 143Subcategories of Extracytoplasmic Substrate Oxidation..................................................... 143Methods of Enzyme Localization ..................................................... 143Oxidations in Which Protons Are from Substrate ..................................................... 144Hydrogen..................................................... 144Photosynthesis..................................................... 144Formate...................................................... 145Methane .................................. , 145Ammonia.................................. 146

Oxidations in Which Protons Are from Water .................................. 147Nitrite.................................. 147Carbon monoxide .................................. 147Reduced sulfur compounds .................................. 147

Sulfide.................................... 147Sulfur .................................. 148Thiosulfate................................. e 148Sulfite ................................. 148

Oxidations in Which Protons Originate from Substrate and Water.............................................. 149Trimethylamine ..................................................... 149Glucose ..................................................... 149

Reactions in Which Protons Are Not Produced from Substrate................................................... 149Iron ..................................................... 149Copper and tin...................................................... 150Uranium ..................................................... 150Manganese...................................................... 150Ferrous c-type cytochromes ..................................................... 150

DISCUSSION ..................................................... 150Requirement for Permease Systems ..................................................... , 150Accumulation of Toxic Substrates or Products ..................................................... 150Prevalence of Gram-Negative Organisms ..................................................... 150Relation to Redox Potentials and Nature of Electron Acceptor ................................................... 150Absence of the Scheme in Oxidizers of Complex Molecules ..................................................... 150Rationale for Cases of Cytoplasmic Oxidation of Simple Reductants ............................................ 151

Cytoplasmic utilization of H ...................................................... 151Involvement of adenine or pyridine nucleotides ..................................................... 151Importance of reaction products to carbon assimilation ...................................................... 151

ACKNOWLEDGMENTS ..................................................... 151LITERATURE CITED ..................................................... 152

INTRODUCTION plasmic, matrix, or stroma side of the membrane, respec-Synthesis of adenosine 5'-triphosphate (ATP) in bacteria, tively. The free energy driving ATP synthesis is generally

mitochondria, or chloroplasts is catalyzed by an adenosine thought to be derived from the transmembrane electrochem-5'-diphosphate (ADP) phosphorylating enzyme on the cyto- ical gradient generated from electron transfer in respiratory

or photosynthetic systems (79, 154, 155, 223). A componentof the electrochemical gradient is the increased concentra-

* Corresponding author. tion of protons on the extracytoplasmic, intermembranet Present address: Department of Microbiology, University of space, or thylakoid cisternae side of the membrane. This

Washington, Seattle, WA 98195. review briefly summarizes known mechanisms for the gen-

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EXTRACYTOPLASMIC OXIDATION OF SIMPLE REDUCTANTS

EXTRACYTOPLASMIC MEMBRANE CYTOPLASMIC

XH2 AH20

X Ard /2022H+ e 2H+

FIG. 1. General scheme for extracytoplasmic oxidation of simplesubstrates. A, Electron carrier.

eration of the proton component of the electrochemicalgradient with special focus on bacteria which oxidize simplereductants [i.e., H20, CH4, CH30H, HCOOH, CH3NH3,(CH3)2NH, (CH3)3N, N02-, NH3, CO, Fe2+, U4+, CU+,Sn2+, Mn2+, or reduced sulfur compounds] as energy source.These bacteria are chemoautotrophic, photoautotrophic, ormethylotrophic. The available data on bacteria which utilizesimple reductants suggest the following new generalization(Fig. 1). In these organisms, the substrate is oxidized on theextracytoplasmic side of the cell membrane. In most casesthe oxidation of substrate results in the production ofprotons from the substrate or water or both in the extra-cytoplasmic compartment. Electrons from the substrate arethen vectorially transported across the cell membrane bymetal redox centers. The electrons eventually reduce aterminal electron acceptor such as dioxygen in a proton-uti-lizing reaction on the cytoplasmic side of the membrane. Thenet result is a proton gradient.

Topology (Fig. 2)

Most chemoautotrophic, photoautotrophic, and methylo-trophic bacteria are gram negative. The major differencebetween gram-positive and gram-negative bacteria is that thelatter contain, in addition to the plasma membrane, a secondmembrane (the outer membrane or lipopolysaccharide layer)external to the peptidoglycan layer. The space between theouter membrane and plasma membrane (including the lumenof invaginations of the plasma membrane) is the periplasm.For the purpose of this discussion an enzyme or redoxcarrier which is soluble in the periplasm or membrane boundwith its substrate-binding site on the periplasmic surface ofthe plasma membrane may be referred to as "extra-cytoplasmic." An enzyme or cytochrome which is soluble inthe cytoplasm or membrane bound with substrate bindingsite on the cytoplasmic surface of the membrane may bereferred to as "cytoplasmic." By analogy, in mitochondriaenzymes located in the intermembrane space, lumen of thecristae, or the outer surface of the inner or cristae membraneare extracytoplasmic; enzymes in the matrix or on the innersurface of the inner or cristae membrane are cytoplasmic. Inchloroplasts, enzymes in the intermembrane space, thylakoidcisternae, or inner surface of thylakoids are extra-cytoplasmic; enzymes in the stroma or on the outer surfaceof thylakoid membranes are cytoplasmic. This topologycorresponds to the scheme adopted by microscopists(229).

Strategies for Establishment of a Proton Gradient (Fig. 3)

Direct "translocation" of HW. The simplest mode of protontranslocation across the cell membrane is observed in thelight-driven proton pump of Halobacterium spp. (23, 46,149, 207); the absorption of light by bacteriorhodopsininduces the protein to transfer protons across the cellmembrane (Fig. 3A). Release or utilization of protons bysubstrate molecules is not involved. Proton pumping activityhas also been observed in cytochrome oxidase of bothmitochondrial and bacterial systems (8, 35, 94, 124, 182, 190,203, 242-244) (Fig. 3A). However, there is debate over thesignificance of this activity (156, 158, 175, 176).

Translocation of H+ through "redox loops." As first pro-posed in the chemiosmotic hypothesis of Mitchell (154), aproton gradient is generated as hydrogens or electrons flowthrough a respiratory chain. Vectorial orientation and alter-nation of electron and H (electron plus proton) carriers(i.e., redox loops) result in the net translocation of protonsacross the cell membrane (79, 154, 155, 223; Fig. 3B). Thescheme most commonly found in organisms which oxidizecomplex organic compounds involves cytoplasmic dehydro-genation of substrate, vectorial translocation of H., theextracytoplasmic release of H ', return of electrons to cyto-plasm by way of membrane metal redox centers (see reviews[29, 79, 88, 156, 157, 223]), and the proton-utilizing reactionof electrons with a terminal electron acceptor such asoxygen. The electron transport components in mitochon-drial, chloroplast, and most heterotrophic bacterial systemsdiffer in detail, but the general scheme of proton transloca-tion is similar.

Cytoplasmic H+-utilizing electron acceptors. The creationof a proton gradient is facilitated by He-utilizing reactions onthe cytoplasmic side of the cell membrane (Table 1).

In the reduction of H+ to H2, H+ is the simplest proton-utilizing terminal electron acceptor. The reduction, in thecytoplasm, of H+ to H2 in anaerobic fermentative bacteriamay contribute to a proton gradient. The extracellular re-duction of H+ would be counterproductive. In Rhodopseu-domonas gelatinosa, which produces H2 during anaerobicgrowth in the dark on CO, H+ appears to be the cytoplasmicterminal electron acceptor (see CO oxidation, Fig. 8C).From the perspective stressed in this review the utilizationof H+ is, in fact, nearly as important a function as theacceptance of electrons.

Also included in this category are reactions in which 02,S042-, CO2, N02-, NO3-, or fumarate is used as terminalelectron acceptors (see reviews [79, 223]). The reduction of02, S042-, C02, and fumarate has long been accepted asoccurring on the cytoplasmic face (223) in many bacteria. Anexception is the fumarate reductase of Clostridium formi-coaceticum (58). Although once thought to be periplasmic(79, 223), the nitrate-reactive site of the dissimilatory nitratereductase enzyme is apparently on the cytoplasmic side

BACTERIA MITOCHONDRIA CHLOROPLAST

FIG. 2. Compartments of bacteria, mitochondria, and chlo-roplasts. c, Cytoplasmic; e, extracytoplasmic.

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142 HOOPER AND DiSPIRITO

EXTRACYTOPLASMIC

A: DIRECT TRANSLOCATION OF H+

1. Bacteriorhodopsin

2. Cytochrome Oxidasecytochromecytochrome

MEMBRANE CYTOPLASMIC

B: REDOX LOOPS-.dt

/

2

C: EXTRACYTOPLASMIC OXIDATIONOF SUBSTRATE XH2

x2H+

FIG. 3. General strategies for generation of a proton gradient. (A) Direct translocations of protons by bacteriorhodopsin or cytochromec oxidase. (B) Redox loops. (C) Extracytoplasmic oxidation. Q, H+ carrier such as coenzyme Q; A, electron carrier.

(102, 110-112, 208). From the perspective of the protongradient, reduction of NO2- is unusual in that it consumesprotons and yet occurs in the periplasm (166, 204, 250). Theadvantage of the periplasmic location of nitrite reductasemay include exclusion of potentially toxic compounds suchas N02- and NO from the cytoplasm.We note that other reactions which are linked directly to

the oxidation of the energy-yielding substrate can be protonutilizing and thus could, in theory, contribute to an energy-linked proton gradient (Table 1). These reactions includemonooxygenase reactions (2H+ + 2e- + XH + 02 -- XOH

+ H2A, see NH3 oxidation below; or H+ + nicotinamideadenine dinucleotide, reduced form [NADH], + XH + 02-k

XOH + NAD+ + H20, see CH4 oxidation below) andreduction of NAD+ by metal redox centers (i.e., H+ + 2e-+ NAD+ -- NADH; see CH4 and H2 oxidation below).

Extracytoplasmic oxidation of simple substrates. In bacteriawhich oxidize compounds of simple structure as an energy

source, the oxidation of substrate often results in productionof protons. Thus, there would be an advantage to having thereaction occur extracytoplasmically (Fig. 1 and 3C). If the

resulting electrons are vectorially transported across themembrane where they reduce a terminal electron acceptor ina proton-utilizing reaction on the cytoplasmic surface, a

proton gradient suitable for driving ATP synthesis wouldresult. The initial extracytoplasmic electron acceptor may beeither a metal or an extracytoplasmic H acceptor whichreleases H+ upon reoxidation at an extracytoplasmic metalcenter. The reoxidation of the electron or H acceptor is, ineffect, analogous to the oxidation of cytochrome c or ubiqui-none in heterotrophic bacteria and mitochondrial systems.The first example of this phenomenon was the extra-cytoplasmic oxidation of formnate reported by Kroger (126).We first encountered this scheme in ammonia oxidation inNitrosomonas europaea (52a, 91, 173) and noted that itoccurred in other bacteria which oxidize simple compounds.If oxidation of energy-yielding substrate were cytoplasmic,the protons produced would contribute to a gradient in thewrong direction. The existence of experimentally observedpH gradients in such organisms would force one to invokevery active proton pumps. The scheme is obviously appro-priate in cases where the precursor or product is not used for

2H±( 1/202

H20

< H20

1/2022H

Aox

Ared'

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TABLE 1. Proton-utilizing terminal electron acceptors and other cytoplasmic proton-utilizing energy-linked reactionsExtracytoplasmic Membrane Cytoplasmic

Terminal electron acceptors 2e- + 1/2 02 + 2H+ H208e- + S042 + 8H + S2- + 4H202e- +NO3 + 2H+ -* N02 + 2H208e- + CO2 + 8H+ - CH4 + 2H202e- + fumarate + 2H+ succinate2e- + 2H+ H,

NH3 + 2H20 -- NO2 + 7H+ + 6e-N20 + 4H20 <- 2NO2- + 8H+ + 6e-1/2 N2 + 2H20 -- NO2 + 4H+ + 5e-

Other H+-utilizing energy-linked reactionsM:onooxygenase 2e- + XH + , + 2H+-> XOH + H20

NADH + XH + 02 +H+ -* XOH + NAD+ + H20NAD+ reductase 2e- + NAD+ + H+-> NADH

other energy-yielding or biosynthetic reactions. Thus, itdoes not apply to most heterotrophic modes of energyproduction. The advantages of this model may include thefollowing: (i) transport of substrate or product is unneces-sary; (ii) toxic accumula-tion of substrate or product isavoided; (iii) redox couples are separated into regions withdifferent pH values on opposite sides of the cell membrane(e.g., Fe2+; see reference 98).

In the present paper we review the evidence for extra-cytoplasmic oxidation of energy-yielding substrates. The listsupports the generalization stated above. There is strongevidence for the scheme in the oxidation of NH2 OH, H2,CH30H, CH3NH2, and HCOOH. Reasonably good indirectevidence exists in the oxidation of Fe2 , S032, S2-, So, andS2032- and of H20, H2, organic compounds, and H2S inphotosynthesis. Evidence for the site of oxidation of NO2-,Sn2+, U4+, or Cu+ is weak or not available. Oxidation ofCH4, CO, HCHO, HCOOH, (CH3)3N, and (CH3)2NH ap-pears to be cytoplasmic. We suggest instances where thereactions might, on future investigation, be found to beextracytoplasmic and we present a rationale for instanceswhich appear to be exceptions.

EXAMPLES OF EXTRACELLULAR OXIDATION OFSUBSTRATE

Subcategories of Extracytoplasmic SubstrateOxidation (Table 2)

The perspective of this review is to draw attention to theproton as a significant product. The reactions can be cate-gorized according to whether the electron-yielding substrateor water or both are sources of extracytoplasmic protons: (i)the substrate may be the source of protons (e.g., H2S -- So+ 2e- + 2H+); (ii) water may be the source of protons (e.g.,HNO2 + H20 -k HNO3 + 2H+ + 2e-); and (iii) theextracytoplasmic enzymatic reaction may not be protonyielding (e.g., Fe2+ -* Fe3+ + e-). In some cases protonsmay be released from water in a non-enzyme-catalyzedreaction [e.g., Fe3+ + 3H20 -* Fe(OH)3 + 3H+].

Methods of Enzyme Localization

We note that it is often difficult to establish whether thecellular location of the site of reaction of substrate with anenzyme is cytoplasmic or extracytoplasmic. Often the con-clusion must be based on the application of several ap-proaches.

Selective release of enzyme after removal of the bacterial

outer membrane is good evidence for extracytoplasmiclocalization (69, 75, 161, 162) (see references 20 and 141 formethods). The complex cell wall structure of gram-negativebacteria makes enzyme localization a more difficult task thanwith gram-positive microorganisms. The first and most prob-lematic step involves the selective release of the outermembrane while leaving the cell membrane intact. The mostcommon procedures for the removal of the outer membraneare based on the initial work of Repaske (192, 193) for theformation of spheroplasts of Escherichia coli and Pseudo-monas sp. with lysozyme-ethylenediaminetetra-acetic acid(EDTA) solutions in the presence of an osmotic stabilizersuch as sucrose (146, 166, 173, 185, 245). In some bacteriathe outer membrane can be removed without lysozyme andin some cases without EDTA. Methods which do not uselysozyme include incubation of washed cells in lithiumacetate or chloride (84, 107, 135), magnesium chloride (37),sodium carbonate (plus EDTA) (17, 75), or sodium chloridefollowed by a sucrose treatment (53, 69, 70, 135, 147). Othertechniques used for the release of periplasmic proteinsinvolve incubation of cells in sucrose-EDTA followed by an"osmotic shock" (86, 103, 161, 162, 164, 173). Unfortu-nately, there is no isolation procedure that works with allgram-negative organisms, so the treatment must be workedout and verified for each case.

Selective release or retention of an enzyme after removalof the bacterial outer membrane is good evidence for extra-cytoplasmic or cytoplasmic location, respectively. How-ever, the result may have alternative explanations. There areinstances where specific cytoplasmic proteins were appar-ently released after the osmotic shock procedure (20, 143).The adhesion sites between the outer and cell membranesmay be involved in this process (19, 20). Lack of release mayindicate a cytoplasmic location but is also consistent withtight binding to membrane or entrapment in the lumen ofmembrane invaginations.Other methods of enzyme localization include the differ-

ential labeling of proteins with compounds such as isethionylacetimidate which penetrate the outer membrane but not thecytoplasmic membrane (96, 116, 117, 152, 160). The compar-ison of the reactivity of cell or spheroplasts with electronparamagnetic resonance labels, inhibitors, or specific anti-bodies has also been utilized (33, 34, 159, 187, 232). Electronmicroscopic studies with specific labeled antibodies or dyeprecipitation have also been used for enzyme localization(43, 126, 150). Because soluble c cytochromes are generallyextracytoplasmic (72, 253), evidence that a c cytochrome isthe natural electron acceptor for an enzyme would suggest

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that the enzyme is also extracytoplasmic. The location of aprotein within a membrane (i.e., transmembrane, inside,outside) has been determined with a number of protein-bind-ing compounds (77, 194). The degree of certainty of locali-zation of specific enzymes will not be extensively discussedhere (see references 20, 43, and 86).

Oxidations in Which Protons Are from Substrate

Hydrogen (Table 2 and Fig. 4 [168]). The oxidation ofdihydrogen is, of course, the simplest possible case of aproton-releasing dehydrogenation. All hydrogenases are Fe/Sproteins and are electron acceptors rather than H * accep-tors. In many instances where H2 is oxidized for energyproduction (as contrasted with reduction of NAD+ forbiosynthesis [28]) the enzyme is extracytoplasmic.

In anaerobic bacteria which derive energy from the oxi-dation of H2 with sulfate, CO2 SI, nitrate, or fumarate aselectron acceptor (2), the hydrogenase is soluble and locatedin the periplasmic space (17, 22, 166, 168, 204, 234). Thelow-potential cytochrome C3 which may be the electronacceptor from H2ase (2, 168) is located in the periplasm (17).Facultative anaerobic bacteria such as Escherichia coli andProteus mirabilis have membrane H2-oxidizing systemswhich donate electrons to a cytochrome (2).Many aerobic H2-oxidizing bacteria have an H2-oxidizing

system which feeds directly into the electron transport chain(2). Extracytoplasmic proton release and vectorial electrontransport associated with the membrane H2ase of Desulfo-vibrio gigas was first proposed by Wood (252). Jones (109)subsequently provided indirect evidence for proton releasefrom H2 in the periplasmic compartment. In E. coli, the

TABLE 2. Extracytoplasmic energy-linked oxidation of simplesubstances

Extracytoplasmic Membrane

H+ from substratehy H2 -> 2H+ + 2e-

H20 --' + + 1/202+ 2e-H2S-2H++ SO + 2e-

H2 2H+ + 2e-organic h-v' 2H+ + organic., + 2e-HCOOH -* 2H+ + CO2 + 2e-NH20H -- 2H+ + HNO + 2e-CH30H - 2H+ + HCHO + 2e-

H+ from H20H20 + N02- 2H+ + N03 + 2e

5H20 + S2032 -* 10H+ + 2SO42- + 8e-3H20 + SO -* 5H+ + HS032- + 4e-

H20 + S032-+ S042- + 2H+ + 2e-

H+ from both substrate and H20H20 + CH3NH2 - 2H+ + HCHO + NH3 + 2e-H20 + glucose -- 2H+ + gluconic acid + 2e-

H+ not produced from substrateFe2+-- Fe3+ + e-

H2S-> 2H++SO+ 2e-Sn2+ Sn4+ + 2e-Cu+ Cu++ + e-Mn2+ Mn4+ + e-

U4+ U6+ + 2e-M2+ + 2H20 -> M(OH)2 + 2H+ +

cytochrome c++ -+ cytochrome c+++ + e-


4 Cytochrome c34H+j3

4 Cytochromem

B2H4 H20

\4~ 2e7/ -co

02

H2* ~ - ---H2

FIG. 4. Extracytoplasmic oxidation of H2. (A) Oxidation of H2produced during oxidation of lactate by D. gigas (modified fromOdom and Peck [168]). (B) Oxidation of H2 produced during COoxidation by D. vulgaris.

membrane H2ase has been localized on the periplasmicmembrane surface (232).Odom and Peck (167, 168) have articulated an interesting

role for H2 in vectorial H transfer in sulfate-reducing bacte-ria grown on lactate (Fig. 4A); H2, which is generated by acytoplasmic hydrogenase, passes across the membrane andis oxidized in a periplasmic H+-producing reaction. In thisinstance H2 can be considered analogous to ubiquinone; it isthe simplest possible form of H * carrier. Evidence for thesignificance of the pathway involves restoration of lactate-dependent sulfate reduction by the addition of periplasmichydrogenase and cytochromes C3 to spheroplasts ofD. gigas(167). Lupton et al. (144) presented indirect evidence thatH2ase and H2 have a different role in this organism, i.e., toappropriately poise the redox potential of cellular electroncarriers during lactate and sulfate-dependent growth in D.vulgaris. The latter authors (144), however, suggested thatthe H2 shuttle operates during CO-dependent sulfidogenicgrowth of D. vulgaris (Fig. 4B).

Species of the nitrogen-fixing bacteria Azotobacter andRhizobium have a membrane-bound, unidirectional, "up-take" hydrogenase. The role of this enzyme is thought to bethe recycling, for ATP synthesis and biosynthesis, of thereducing equivalents in H2 produced by the futile cycles ofnitrogenase (2, 67, 123). Whether the biological oxidant is anelectron or H * acceptor is unclear. In Azotobacter vinelandiihigh-potential acceptors (123) or ubiquinone (250) has beenimplicated. In Rhizobium japonicum, b cytochromes (59) ora high-potential flavin (165) is a possible electron acceptor. Ifthe reaction produces protons (i.e., H2 -- 2H+ + 2e-) it

might logically be extracytoplasmic. The H2-reactive site ofthis enzyme has not been localized in periplasm or cyto-plasm.With H2 oxidation by D. gigas coupled to reduction of

nitrite on what was assumed to be the periplasmic membranesurface (18), it was concluded that the proton gradient wasachieved exclusively through electron transfer-coupled trans-location since the two periplasmic reactions would not leadto a net gain of protons (i.e., 3H2 -* 6H+ + 6e-; HNO2 +

6e- + 6H+ -- NH3 + 2H20).

Photosynthesis (Fig. 5 and Table 2). From the perspectivepresented here, oxygenic photosynthesis may be viewed as aproton-releasing, light-energy-dependent H20 dehydroge-nase. In cyanobacteria and chloroplasts it is clear that thesite of oxidation of H20 is in the membrane. To ourknowledge the reaction has not been topologically localizedin cyanobacteria. Thus, we are forced to rely on analogy

C02

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FIG. 5. Bacterial photosynthesis during (a) cyclic and (b) noncyclic photophosphorylation. For simplicity the flow of only one electron isshown. XH, Substrate electron (and proton) donor; Q, quinone.

with chloroplasts. In chloroplasts the evidence favors local-ization on the internal surface of thylakoids (3, 9, 10, 51, 114,246) (i.e., the extracytoplasmic surface; Fig. 2). As such itfits the generalization reviewed here.

Oxidation of substrate (H2S, S°, S2032-, H2, or organiccompounds) during anoxygenic photosynthesis also appearsto be extracytoplasmic (Fig. 5), although the evidence in thiscase is indirect. The membrane topology of the photosyn-thetic reaction center has been determined in several bacte-ria (16, 42, 183, 231, 240, 257). In the organisms examined,one or more of the reaction center subunits (H, M, or L) areexposed at the periplasmic surface of the membrane. Duringcyclic photophosphorylation soluble and membrane c-typecytochromes (or multiple membrane-associated c-type cyto-chromes) have been shown to reduce the reaction center (47,48, 63, 68, 113, 172, 188, 200, 233, 236). The solublecytochrome (cytochrome c2) has been localized in the per-iplasm ofRhodopseudomonas sphaeroides and R. capsulata(187).The cellular location of the enzymes involved in the

oxidation of electron donors for anoxygenic photosynthesisand CO2 reduction has received less attention. In green andpurple sulfur bacteria type c cytochromes are the primary orsecondary electron acceptors from thiosulfate cytochrome creductase (130-132, 205, 206). A flavocytochrome c is appar-ently involved in the oxidation of sulflde or thiosulfate (73,131, 205, 206). These type c cytochromes are also rapidlyphotooxidized and are the electron donors to the reactioncenter (219). In most cases, two type c cytochromes areinvolved; one of the two is soluble and therefore probablyperiplasmic. Analogy with the location of c cytochromes incyclic photophosphorylation also suggests that these cyto-chromes are periplasmic. Extracellular oxidation of reduc-tant is also indicated by the extracellular location of sulfur(i.e., outside the cell) in Chlorobiaceae or within membrane-bound SI granules (i.e., extracytoplasmic) in Chromatiaceaeduring H2S oxidation (184, 225).Formate (Table 2). Kroger (126) first suggested that extra-

cytoplasmic oxidation of formate coupled to vectoral elec-

tron transfer to fumarate was a basis for the generation ofproton gradient in Vibrio succinogenes. Studies of dyeaccessibility have suggested that formate oxidation is peri-plasmic (83, 125) or cytoplasmic (74) in E. coli. Enzymerelease after removal of the outer membrane (126) haslocalized formate dehydrogenase in the periplasm of V.succinogenes, D. gigas, and E. coli. In D. gigas cytochromeC553 may be the initial electron acceptor (166).Formate oxidation in methylotrophs is discussed below.Methane (Table 3 and Fig. 6). Methane is oxidized to CO2

with methanol, formaldehyde, and formate as intermediates(12, 87). Methane monooxygenase catalyzes the reaction 02+ NADH + H+ + CH4 -- NAD+ + CH30H + H20 (49).The enzyme occurs in both soluble and particulate form (12,87). Physical location of the enzyme as cytoplasmic orextracytoplasmic has not been determined. Noting thatNADH is a substrate, it is generally assumed that thereactions are cytoplasmic. This location is also consistentwith the fact that they are proton utilizing (Table 3).Reducing equivalents from the formaldehyde, formate,

and perhaps methanol dehydrogenase reactions are utilizedin methane monooxygenase, in reduction of NAD+ forbiosynthetic reactions, or in electron transport leading toATP synthesis.Although the prosthetic group of methanol dehydrogenase

is a novel quinone coenzyme (pyrroloquinoline quinone) theenzyme utilizes a soluble cytochrome c as electron acceptorand is therefore a proton-yielding dehydrogenase (21). Lo-calization studies have shown that methanol dehydrogenase(8, 32, 116, 117) and the electron acceptors cytochromes CLand possibly cytochrome CH (21, 32, 108, 191) are in theperiplasm (8, 116). Thus, methanol oxidation clearly fits thegeneralization described here.Formaldehyde and formate dehydrogenase occur in forms

which use either NAD+ or dyes as electron acceptors (106,148). Substrate oxidation is proton yielding and could logi-cally be periplasmic (H20 + HCHO -* 2H+ + 2e- +HCOOH; HCOOH -- 2H+ + 2e- + C02). Positing cross-membrane translocation of electrons to the proton-utilizing

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TABLE 3. Oxidation of methane, carbon monoxide, sulfur, and trimethylamine involving both cytoplasmic and extracytoplasmiccompartments

Compound Extracytoplasmic Membrane Cytoplasmic

Methane H+ + CH4 + NADH + 02 --+CH30H + H20 + NAD+CH30H -> 2H+ + HCHO + 2e- H20 + HCHO -* 2H+ + HCOOH + 2e-

HCOOH -* 2H+ + CO2 + 2e-

Carbon monoxide H20 + CO -- 2H+ + 2e + CO2

Sulfur 5H20 + S2032- 1OH+ + 2SO42- + 8e-3H20 + S-- 5H+ + HS03- + 4e-

H20 + so32- s042- + 2H+ + 2e- P043- + H20 + S032- + AMP--* 2H+ + S042- + ADP + 2e-

Trimethylamine H20 + (CH3)3N -* 2H+ + (CH3)2NH + 2e- + HCHO

H20 + CH3NH2 -2H+ + HCHO + NH3 + 2e- H20 + (CH3)2NH --2H + CH3NH2 + 2e + HCHOH20 + HCHO -O 2H+ + HCOOH + 2e-HCOOH --* 2H+ + CO2 + 2e-

reduction of NAD+ (H+ + 2e- + NAD+ -- NADH), theaction of a dehydrogenase located in the periplasm wouldgenerate a proton gradient in both reactions. This seems notto be the case with the NAD-linked formaldehyde (116) andformate (108) dehydrogenases; because they are soluble anduse NAD, they are probably cytoplasmic. The topologicallocation of the dye-linked enzymes is unknown.To summarize, in the methylotrophs the proton-utilizing

methane monooxygenase may be cytoplasmic and the ener-gy-linked proton-yielding oxidation of methanol is peri-plasmic. Thus, CH30H functions as a transmembrane Htransporter. In contrast, NAD-linked oxidations of formal-dehyde and formate, which produce reductant for methanemonooxygenase, biosynthesis, or electron transport leadingto ATP synthesis and C02 for carbon assimilation, areapparently cytoplasmic.Ammonia (Fig. 7; reviewed in reference 90). The best

EXTRACYTOPLASMC MMBRANE CYTOPLASMIC

CH4

---CH30H

,.assimiilation

- - - v HCHO

H20 NAD+

< NADH, H+

2H+

H20

2H5,

- - -vHCOOH2 NAD5

C0 NADH+H4

- -_ assimilationFIG. 6. Oxidation of methane by methylotrophic bacteria. Al-

though the significance in vivo is not known, the extracytoplasmicmethanol dehydrogenase can also oxidize formaldehyde (21) asdepicted in reaction a'.

evidence indicates that NH3 is oxidized to NH20H by anoxygenase. Hydroxylamine is then oxidized to HNO(NH20H -* HNO + 2H+ + 2e-) by the heme P460 center ofthe multiheme enzyme hydroxylamine oxidoreductase. Elec-trons pass through c hemes of the enzyme (92), possibly totwo soluble cytochromes, C554 and c552 (254), and then,presumably, to a membrane-terminal oxidase. The hydroxy-lamine-oxidizing enzyme (173) and cytochromes c554 and c552are periplasmic (52a, 91) in keeping with the general schemeproposed here.The mechanism of oxidation of HNO by the periplasmic

hydroxylamine oxidoreductase is less clear. Recent evi-dence suggests the reaction HNO + H20 -- HN02 + 2H+ +2e- (11) although the reaction HNO + 1/202 -- HN02 hasnot been clearly ruled out. The former reaction wouldcontribute additional periplasmic protons.Whether oxidation of ammonia is by a dioxygenase (2NH3

+ 02 -- 2NH20H + H20) or a monooxygenase (NH3 + 2H++ 2e- + 02 -* NH20H + H20 [226]) is unknown. If amonooxygenase is involved, the coreductant is probably ametal center (rather than an H donor such as NADPH) withelectrons originating from oxidation of NH20H in the per-iplasm. Because protons are utilized, there would be some

EXTRACYTOPLASMIC MEMBRANE CYTOPLASMICNH3 I

HN02NC'

1/2 02\H20 -'

c

2H+HN02

H20

02, 2H+

FIG. 7. Oxidation of ammonia by bacteria. Reactions b and c ori are known to be extracytoplasmic. It is not known whetheroxidation of ammonia or nitroxyl occurs by reaction a or a' or c orc', respectively. The compartment of ammonia oxidation isunknown.

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A

extra-cytoplasmic membrane cytoplasmic

a///l CO2

2Hs

B H20

co.

002

2H+

cH20

co.

2H

2e

co

'H20

2H+

H2

FIG. 8. Oxidation of carbon monoxide by bacteria. Possiblecytoplasmic (A) or extracytoplasmic (B) locations. (C) Possiblescheme in R. gelatinosa.

logic in having a monooxygenase-catalyzed reaction on thecytoplasmic side of the membrane.

Oxidationg in Which Protons Are from Water

Nitrite (Table 2). In the oxidation of N02- to NO3- byNitrobacter sp., H20 is apparently the source of oxygen (7,89, 129). The reaction is therefore a proton-yielding dehy-drogenase (H20 + N02-* 2H+ + NO3-). A mtembrane-bound nitrite mammnalian cytochrome c reductase fromNitrobacter sp. contains c and a hemes (120) in keeping witha role of electron (rather than H) removal during oxidation.The topological location of the NO2 oxidation site has notbeen determined. Based on the generalization surveyedhere, location on the periplasmic surface is predicted. Soneet al. (203) report that purified Nitrobacter agilis cytochromeoxidase which has been reconstituted in vesicles lacksproton pumping activity. If this is also the case in vivo, theneed for extracytoplasmic location of NO2- oxidation ap-pears very likely.Carbon monoxide (Fig. 8). The aerobic CO-oxidizing che-

molithoautotrophic bacteria are able to use the oxidation ofCO for ATP production, reductant for biosynthesis, andcarbon source (reviewed in references 28 and 151). COdehydrogenase is a flavin, Mo-, Fe-, and S-containing dyereductase (151). The proton-yielding reaction H20 + CO -.

CO2 + 2e- + 2H+ could logically be periplasmic andcoupled to transmembrane electron trahsport (Fig. 8B). Theenzyme is found in both soluble and membrane forms. Thedistribution between cytoplasm and periplasm has not beendetermined for the soluble form. Immunolocalization byelectron microscopy indicates that the antigenically activeregion of the membrane form is on the cytoplasmic surface(150). If the enzyme is transmembrane, the proton-yieldingCO oxidation site could still be localized on the outersurface. Assuming that, in keeping with the best evidenceavailable, CO oxidation is cytoplasmic, it represents anapparent exception to the generalization summarized here.In fact, since oxidation of CO may be coupled at the level ofubiquinone (122), and assuming that ubiquinone is reoxi-dized on the periplasmic surface, the gain of periplasmicprotons would be the same with CO dehydrogenase in eitherthe cytoplasm or the periplasm (Fig. 8A). The location of COoxidase in the cytoplasm may have to do with C assimila-tion. As much as 16% of the CO carbon is assimilated byCO-oxidizing autotrophs (151). Autotrophs can apparentlyencounter difficulty in production of adequate levels ofcytoplasmic CO2 for carbon fixation (103a). This require-ment is indicated by the presence of inducible carbonicanhydrase and CO2 uptake systems (24, 41, 78, 169). Thedesirability of a cytoplasmic C02-producing system mayhave led to location of CO oxidation in the cytoplasm.R. gelatinosa will grow anaerobically in the dark with CO

as sole energy and carbon source (228, 229). CO2 and H2 areproduced in equal quantities so the approximate overallreaction appears to be xCO + yH20 -) mC02 + mH2 +nCHO. CO oxidation is catalyzed by a membrane dehydro-genase able to use a low-potential (-548 mV) hydrogenacceptor. The question of the mechanism of ATP generationin the absence of common electron acceptors has puzzledinvestigators. A plausible mechanism which utilizes theproton as electron acceptor is shown in Fig. 8C. In this caseH+ would be the cytoplasmic proton-utilizing terminal elec-tron acceptor. Unless the CO dehydrogenase from thisspecies can reduce an H * carrier of low enough potential toreduce H+, the proton-yielding oxidation of CO wouldlogically be extracytoplasmic.The energy-yielding CO dehydrogenases mentioned above

are distinct from the Ni-containing CO dehydrogenasesinvolved in acetate catabolism or anabolism or both of C1compounds in the methanogenic (71, 127) and acetogenic(93) bacteria.Reduced sulfur compounds (Table 3 and Fig. 9). The

oxidation of reduced sulfur compounds as a sole or co-en-ergy source occurs in several obligate and facultative che-moautotrophs and in mixotrophic bacteria (31, 104, 128,195-197, 210). The details of the complex mechanism ofsulfur oxidation is beyond the scope of this review and willonly be considered briefly here (see references 6, 116, 118,120, 121, 181, 195, 210, and 216). We only consider theoxidation of S2-, So, S2032-, and S032- by the thiobacillisince the enzynmes in these oxidation have been partiallycharacterized and the role of polythionites as intermediatesis in doubt (116, 120).

Sulfide. The possibility that oxidation of sulfide occursextracytoplasmically (Fig. 9) is suggested by the precipita-tion of elemental sulfur under certain culture conditions(116, 177, 218) in the growth medium of some thiobacilli oron membrane proteins in cell-free extracts (199). The site ofoxidation of sulfide to elemental sulfur (sulfide oxidase) hasnot been demonstrated and the enzyme has not been iso-lated. Intracellular sulfur granules are observed in the mixo-

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3H20 Sulfite 6H+,6e& Cytochrome b

Oxias see,222-dtae2s s 3~~~~~~~~~~~~~~~~~~~~~

Sulf__it2e pAM APSSulfide H2 Slytochrome c Reductase

2nWn 2e3 ;W -p2D

Oxidase 2e Oxidoreductase 2eso - 5Q2- APiN 4P1~~~4ADP

SulfurH20 02 SCo mee- S sociatSulfurylase

FG9.Pssilf arraneeto h nye novdi h oxidtiohromerdce sufrcmons 4hcem sacmoieoh

Oxygenase ADP2HW 2e

non-enzymic 20 -- - S203Thiosulfate 51-20

Cytochrome QOxidorecductase Rhodanese

2 H2010H+ 8e 02 2

2S0- 5034H+ 'Transient Membrane

soj- ~~~~~-Associated Sulfur Sol'7/7/ C~~~~So)FIG. 9. Possible arrangement of the enzymes involved in the oxidation of reduced sulfur compounds. The scheme is a composite of the

reactions catalyzed by several different species of thiobacilli.

trophic H2S-oxidizing bacteria Beggiatoaceae and Thiotrix(209, 210). However, the sulfur inclusions are surrounded bya membrane (209) (i.e., are extracytoplasmic), leading to theproposal (210) that the oxidation in Beggiatoa is extra-cytoplasmic.

Sulfide is also oxidized directly to sulfite: S2- + 3H20 >S032- + 6H+ + 6e-.The enzyme involved (siroheme sulfite reductase) has

been purified in Thiobacillus denitrificans (200). It is asoluble enzyme, but its cellular location has not been deter-mined. Because the reaction releases six protons fromwater, we propose that the enzyme is periplasmic (Fig. 9).

Sulfur: 5° + 02 + H20 -- S032- + 2H+. Elemental orcolloidal sulfur is insoluble. It is necessary that T. thio-oxidans cells have direct physical contact with elementalsulfur before sulfur oxidation can occur (207, 239). There-fore, sulfur is oxidized at the cell surface, dissolved in thecell membrane, or taken up as elemental sulfur by the cells.Although the transport of sulfur into T. thiooxidans inmembrane-bound vesicles has been proposed by Karavaikoand Pivovarova (115, 186), this observation is of question-able significance to the specifics of sulfur oxidation since thesame vesicles have been observed in iron-grown T. fer-rooxidans (52). The authors (186), in fact, propose thatthe oxidation occurs on the outer surface of the cell mem-brane.The sulfur-oxidizing enzyme has been partially purified in

a number of thiobacilli (36, 201, 218, 217, 222) and shown tobe an oxygenase (215) which requires reduced glutathionefor activity (36, 201, 214, 217) in the soluble form but notwhen associated with the membrane fraction (1). The local-ization of sulfur oxygenase has not been determined. Theextracytoplasmic location of sulfur oxygenase would elimi-nate the necessity to transport an insoluble substrate (S)and would result in the extracytoplasmic release of twoH+/S.

Thiosulfate. All species of thiobacilli studied oxidize thio-sulfate to sulfate. Two different mechanisms of oxidation areobserved depending on the species of thiobacillus. The firstinvolves the initial splitting of the S-S bond by rhodanesewith the formation of sulfite and elemental sulfur (36, 199,220). Rhodanese is a soluble enzyme (178-180), but itscellular location is unknown. Sulfur is found as a membrane-associated sol and subsequently converted to s042- (199).The nature and location of the latter reaction are alsounknown. In keeping with the theme of this review it islogical to suggest that the protons and sulfate produced inthe reaction are released on the extracellular membranesurface.The second mechanism involves thiosulfate:cytochrome c

oxidoreductase. The proposed intermediates (So and S032-)are not detected (138) in the reaction. In T. versutus (for-merly Thiobacillus sp. strain A2 [80]), thiosulfate:cyto-chrome c reductase has been purified and is a solublefour-component complex (137, 139). The same activity wasobserved in the membrane fraction of T. novellus (170, 171).The enzyme has not been localized. We suggest that it mayprove to be extracytoplasmic since the oxidation of S2032-releases 10H+(S2032 + 5H20 -* 2SO42 + 10H+ + 8e-),whereas 8e- and therefore 8H+ are presumably consumedby the cytoplasmic terminal oxidase. That oxidation ofS2032- is extracytoplasmic is also suggested by the fact thatthiosulfate electrons enter the electron transport chain at thecytochrome c level (4, 5, 120, 139).

Sulfite. Two mechanisms also operate in the oxidation ofsulfite depending on the species of thiobacillus. The adeno-sine 5'-monophosphate (AMP)-dependent sulfite oxidationpath has been observed in T. thioparus (178-180) and T.denitrificans (27). Because it uses the small solublg substrateAMP, it seems likely to be cytoplasmic. The reactioninvolves a substrate-level phosphorylation, where APS isadenine phosphosulfate:

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2-APS-reductases032 + AMP - * APS + 2e

APS + P043 ADP-sulfurylase ADP + S042P adenylate kinase2ADP - AMP + ATP

On the other hand, sulfite oxidase (sulfite cytochrome creductase) (138, 224, 255) seems likely to be extra-cytoplasmic in bacteria. Interestingly, in rat liver mitochon-dria, sulfite oxidase is observed in the intermembrane space(40, 174). The enzyme has been purified from T. novellus(224, 255) and T. versutus (138, 140) and is a solubletwo-component enzyme. The reaction results in the releaseof two protons and is coupled at the level of cytochrome c(238, 251): S032- + H20 - S042- + 2H+ + 2e-. Theinvolvement of cytochrome c further suggests that theenzyme is periplasmic.

Oxidations in Which Protons Originate from Substrateand Water

Trimethylamine (reviewed in reference 12 [Table 3]). In theanaerobic hyphomicrobia, the oxidation of trimethylamineto formaldehyde and methylamine is catalyzed by trimethyl-amine dehydrogenase and dimethylamine oxygenase (Fig.7). The two enzymes are cytoplasmic (32, 116). Methylaminepasses to the periplasm (acting as an H * carrier) where it isoxidized to formaldehyde by mnethylamine dehydrogenase(32, 116). The latter enzyme is found in many species ofmethylotrophs (12). The reaction is proton releasing(CH3NH2 + H20 -O HCHO + NH3 + 2e- + 2H+). Theenzyme contains a quinone but, by analogy with methanoloxidation, is thought to reduce c cytochromes. Oxidation ofmethylamine fits the scheme of extracytoplasmic protonrelease reviewed here. Formaldehyde, on the other hand, isoxidized cytoplasmically (see above, "Formate"). The evo-lutionary rationale for locating trimethyl- and dimethylaminedehydrogenases in the cytoplasm may be that they produceHCHO used for carbon assimilation or that it is desirable tocouple the oxidation to NAD as the electron acceptor orboth.

Glucose (Table 2). Glucose uptake is diminished in mutantstrains of Pseudomonas aeruginosa lacking glucose dehy-drogenase, suggesting that the membrane-bound enzymeglucose dehydrogenase is extracellular and that glucose maybe oxidized before uptake (152). The enzyme containspyrroloquinoline quinone and utilizes redox dyes (62). Ifcoupled to a metal electron acceptor, the reaction would beproton yielding (glucose + H20 -O gluconic acid + 2H++2e-) and could, in theory, contribute to an energy-transduc-ing proton gradient. Existing evidence suggests that thereaction is energetically significant (89a).

Reactions in Which Protons Are Not Produced fromSubstrate (Fig. 10)

Iron. The oxidation of ferrous iron (and possibly U4+,Cu+, Sn2+, and Mn2+) is unique in that there are no protonsproduced in the enzyme-catalyzed reaction: (i) Fe2' -* Fe3++ e. The ferric iron formed undergoes abiotic hydrolyticreactions which result in the net release of protons on theextracytoplasmic side. The predominant reaction at pH 2.0is (ii) Fe3+ + H20 -* Fe(OH)3 + 3H+. Several groups ofbacteria oxidize ferrous iron as the sole source of energy (76,81, 142, 189, 249). However, the following discussion willdeal only with iron oxidation by T. ferrooxidans.

Addition of Fe2+ to T. ferrooxidans results in an initialextracellular pH increase followed by a net pH decrease inthe medium (13). It has been suggested (13) that the initialincrease in extracellular pH is a result of proton uptake(perhaps by the energy-linked ATPase; 13) followed byproton utilization by the terminal oxidase (13). The second-ary decrease in extracellular pH is attributed to the abiotichydrolysis of Fe3+ (reaction ii).The membrane-associated electron transport components

involved in iron oxidation include the enzyme iron oxidase(25, 39, 211, 237, 256), type c cytochrome(s) (25, 98, 99, 101,256), and the terminal oxidase cytochrome a1. The solubleelectron transport components include c-type cytochrome(s)and a blue copper protein, rusticyanin (39, 44, 45, 98). Aniron cytochrome c reductase or iron oxidation-stimulatingfactor has been reported by several authors (25, 50, 211-213,256). The enzyme has not been purified; its existence isbased on its ability to stimulate the rate of oxidation of Fe2+and reduction of cytochrome c. The existence of ironcytochrome c reductase is questionable (98), and the activitymay in fact be catalyzed by partially purified rusticyanin.

Several observations indicate that ferrous iron is oxidizedextracytoplasmically in T. ferrooxidans (Fig. 10). Experi-ments with cell-free membrane preparations indicate thatonly "right-side-out" vesicles have significant iron oxidaseactivity (25, 26, 39). The interaction of cytochrome c,rusticyanin, and Fe3+ but not cytochrome a1 with extra-cytoplasmic paramagnetic probes suggests that the formerelectron transport components are extracytoplasmic (98).The extracytoplasmic oxidation of iron is also inferred fromthe fact that iron would be rapidly autooxidized at cytosolicpH values (pH 6.5) and that ferric iron formed at this pHwould be insoluble. Other indirect evidence includes (i) thelow concentrations of iron associated with the cell (53) and(ii) the fact that electrons are coupled from iron to theelectron transport chain at the level of soluble cytochrome c(39, 55, 98). Many authors have proposed that oxidation offerrous iron is extracytoplasmic (38, 39, 45, 61, 98, 100).The lack of outer membrane and cell wall in the two

iron-oxidizing Sulfolobus sp. (30, 31) makes the existence ofa freely soluble periplasmic enzyme system unlikely. Theauthors propose that the iron oxidation site is on theperiplasmic surface side of the cell membrane.

EXTRACYTOPLASMIC

2FeOH2 ,

nor

2 H-iO

2H+

2 rusticyanin red 2 rusticyaninox

mif it

MEMBRANE CYTOPLASMIC

'iron oxidase'

pH=2 V/I/u/////A pH-SFIG. 10. Proposed scheme for the oxidation of ferrous iron by T.

ferrooxidans; modification from Ingledew (98).

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Copper and tin. T. ferrooxidans has also been reported tooxidize cuprous and stannous ions (97, 136, 163). Thebiological significance of the oxidation of these metals is stilluncertain (see reference 57). If verified, an oxidation mech-anism similar to that of ferrous iron seems likely.Uranium. In addition to ferrous iron (see above) and

reduced sulfur compounds (see above), T. ferrooxidans willoxidize U4+ to U6+. It is uncertain whether, in T. fer-rooxidans, U4+ is oxidized to U6+ (54-56, 205) directly or

whether soluble iron acts as a redox carrier (82, 147). Ineither case the same electron transport components are usedfor oxidation of both iron and uranium (55, 56). In addition tothe evidence presented for the extracytoplasmic oxidation offerrous iron, other indirect evidence of the extracytoplasmicoxidation of uranium includes (i) the low levels of uranium inthe cytoplasmic fraction of cells actively oxidizing uranium(53, 227) and (ii) the toxicity of uranium.

Manganese. Autotrophic growth of a Pseudomonas sp. hasrecently been achieved in chemostat cultures (K. Nealson,personal communication). The oxidation of Mn2+ has beenshown to be catalyzed in several unrelated bacteria (14, 59,60, 66, 105, 153). The mechanism of oxidation of manganeseby bacteria is not well understood. It may occur by enzy-matic or nonenzymatic mechanisms (14, 59, 60, 65, 66, 105,153, 248) or both. The mechanism appears to differ betweenspecies and the oxidation is not always coupled to ATPformation (66). The enzymes involved in oxidation of Mn2+have not been characterized or localized. In at least twomarine isolates oxidation of Mn2+ appears to be coupled atthe level of cytochrome c (14). Ehrlich (66) has proposedthat the oxidation is extracytoplasmic. The extracytoplasmicprecipitation of manganese compounds, the low concentra-tions of Mn2+ associated with the cells (59), and the knowntoXicity of Mn2+ are consistent with the extracytoplasmiclocation.

Ferrous c-type cytochromes. We note that the most ubiq-uitous example of a non-proton-releasing extracytoplasmicoxidation is that of ferrous c cytochromes in bacterial or

mitochondrial electron transport (253) and bacterial (253) or

chloroplast (48) photosynthesis.

DISCUSSION

In this paper we have briefly reviewed the site of substrateoxidation (whether it is extracytoplasmic or cytoplasmic) inmicroorganisms which utilize the oxidation of simple reduc-tants as a source of energy. The mechanism by which all or

a portion of the proton gradient is generated in many of theseorganisms can be summarized as follows: (i) extra-cytoplasmic oxidation of the substrate with the release ofprotons from the substrate or water or both; (ii) transmem-brane movement of electrons through electron carriers (usu-ally metal centers); and (iii) cytoplasmic reaction of sub-strate electrons with cytoplasmic protons and a terminalelectron acceptor. In this discussion we point out severaladvantages of the extracytoplasmic location of the oxidationand suggest a rationale for the exceptions.

Requirement for Permease SystemsAn advantage of the extracytoplasmic oxidation of simple

reductants is the elimination of the necessity to transport thesubstrate into and the product out of the cell. These trans-port processes would require energy. This may be of specialimportance wheh the substrate is charged (NO2-, S032-S2032, Fe2+, U4+, Cu+, Sn2+, S2) or insoluble (S°) andless important with uncharged substrates (H20, H2, CO,

NH3, CH30H, HCHO) which may diffuse into or across themembrane in a passive manner.

Accumulation of Toxic Substrates or ProductsMany of the compounds oxidized or produced by these

organisms are toxic (H2S, Fe2+, U4+, Cu2+, Sn2+, Mn2+,N02-, HCHO) at substrate concentrations and some prod-ucts such as Fe3+ and SI are insoluble at neutral pH values.The oxidation of these compounds extracellularly wouldminimize toxic effects.

Prevalence of Gram-Negative OrganismsMany of the periplasmic enzymes and electron acceptors

involved in the bacterial oxidation of simple substances aresoluble. The need to contain these proteins probably ex-plains why these mechanisms evolved almost entirely ingram-negative bacteria. Indeed, this may extend to the outermembrane of mitochondria and chloroplasts which acts as abarrier to prevent the loss of soluble c cytochromes.

Relation to Redox Potentials and Nature of ElectronAcceptor

For many of the simple substrates discussed here theperiplasmic site of oxidation may have been partially im-posed by the oxidation-reduction potential. The redox po-tentials of many of the substrates are approximately equal toor higher than the redox potential of ubiquinone (Table 4).Thus, instead of the hydrogen acceptor ubiquinone, they usea higher potential electron acceptor (a metal or heme). Theresulting reactions are proton yielding. If the reaction werecytoplasmic, an acidification of the cytoplasm would occur.Hence the extracytoplasmic location for substrate oxidationbecomes obligatory unless vigorous pumping of protonsduring electron transport is invoked.

Regardless of the substrate redox couple, substrate oxida-tions are, in fact, usually coupled to the electron transportchain at the level of cytochrome c.

Table 4 compares the calculated value of A"4+ withcytoplasmic substrate oxidation with the corresponding valuefor extracytoplasmic oxidation of substrate. The value ofAH' was calculated based on the assumption of 2H+/e-transported in the first two redox loops (241, 244). Theterminal oxidase (third redox loop) was assumed to consume1H+/e- (2e- + 2H+ + 1/202 -- H20). In addition to its rolein the third redox loop, cytochrome c oxidase in somebacterial systems (8, 182, 203) has been observed to act as aproton pump with a value of 0.25 to 1H+/e-. This value ofprotons pumped by the terminal oxidase has not beenincluded in the AH+ values in Table 4. If included, the valuesin both cytoplasmic and extracytoplasmic columns wouldincrease by 0.25 to 1H+/e-. For a more detailed considera-tion on the bioenergetics of chemolithotrophic and methylo-trophic bacteria the reader is referred to recent reviews (12,28, 38, 45, 98, 118-120, 181, 216, 247).

Absence of the Scheme in Oxidizers of Complex Molecules

Heterotrophic organisms have presumably not utilizedthis scheme (but instead translocate H from cytoplasm viaubiquinone) because the precursor, substrate, or product ofthe energy-yielding reaction is often used for other anabolicor catabolic reactions. Location of these oxidative steps inthe periplasm would not only require outward transport ofmetabolites, but also risk loss of substrate or productthrough the outer membrane.

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TABLE 4. Calculated AH', coupling sites, and standard redox potentials for reactions catalyzed by chemoautotrophic andmethylotrophic bacteria

AH+

Reaction E0' (V)- Entry into electron Extra-transport chain Cytoplasmic cytoplasmicreaction reaction

CO + H20 -+ CO2 + 2H+ + 2e- -0.54a Quinone 4 4S032- + H20 - S042- + 2H+ 2e- -0.52b 1. Cytochrome c 0 4

2. AMP/cytochrome c 3HCOOH + 1/2 02 -* CO2 + H20 + 2e- -0.46c NAD+ 8HCOH + 1/2 02 - HCOOH + 2e- -0.45c NAD+ 8H2-- 2H+ + 2e- -0.41d 1. Cytochrome c 0 4

2. NAD+ 8NADH2 -- NAD+ + 2H+ + 2e- -0.32c 8H2S -+ So + 2H+ + 2e- -0.28d Cytochrome c 0 4S2032- + 5H20 -* 2SO42- + 1OH+ + 8e- -0.24e Cytochrome c -2 18So + 3H20 - S032- + 6H+ 4e- -0.2e Cytochrome c -2 10CH30H -- HCOH + 2H+ + 2e- -0.18c Cytochrome c 0 4CH3NH2 + H20 -* HCOH + NH3 + 2H+ + 2e- Cytochrome c 0 4S2- + 3H20 - S032- + 6H+ + 6e -0.l2b Cytochrome c 0 12Mn2+ + 2H20 -- MnO2 + 4H+ + 2e- -0.05f Cytochrome c -2 6Ubiquinone -- ubiquinone0x + 2H+ 0.069 6NH20H + 1/202 -) NO2- + 3H+ + 4e- 0.07h Cytochrome c 1 7Sn2+ _. Sn4+ + 2e- 0.15' Cytochrome c 2 2Cytochrome c" -- cytochrome cll + e- 0.25C 1 1U4+ U6+ + 2e- 0.33' Cytochrome c 2 2NH3 + H20 + 1/202 -* N02- + 5H+ + 6e- 0.34b Cytochrome c 1 11N02- + H20 -- N03- + 2H+ + 2e- 0.43b Cytochrome c 0 4Cu+ Cu2+ + e- 0.52' Cytochrome c 1 1Fe2+ Fe3+ + e- 0.77' Cytochrome c 1 1

a References are: (a) Meyer and Schlegel (151); (b) Thauer et al. (223); (c) Anthony (12); (d) Kelly (119); (e) Wheelis (247); (f) Latimer (134); (g) Urban andKlingenburg (230); (h) Aleem (6); (i) Huheey (95). See text for calculations and entry into electron transport chain.

Rationale for Cases of Cytoplasmic Oxidation of SimpleReductants

Cytoplasmic utilization of H+. The extracytoplasmic loca-tion of H+-producing substrate oxidation contributes to theproton gradient. In keeping with this logic, portions ofenergy-yielding reaction sequences would, in theory, becytoplasmic because they are H+ utilizing (e.g., 2e- + CH4+ 2H+ + 1/202 -* CH30H + H20). It may be argued thatthe location of methane monooxygenase in the cytoplasm isbest explained by the fact that NAD(P)H is a reactant andthat NAD would not be expected in the periplasm because ofthe potential for leakage through the outer membrane. Wenote, however, that production of CH30H in the periplasmwith cytoplasmic NADH as reductant could, in theory, havebeen possible with a transmembrane enzyme: 2H+ + CH4 +1/202 + 2e - H20 + CH30H (periplasm); NADH --

NAD+ + H+ + 2e- (cytoplasm). The disadvantage of thisarrangement is that the cytoplasm would be acidified. Thusthe resulting distribution of protons rather than the involve-ment of pyridine nucleotide may explain the location ofreactions such as methane monooxygenase (Tables 1 and 3).

Involvement of adenine or pyridine nucleotides. One wouldexpect the oxidation of thiosulfate by the adenosinephosphosulfate pathway to be cytoplasmic since location inthe periplasm would risk loss of adenine compounds throughthe outer membrane and, further, impose the need to moveATP across the plasma membrane into the cytoplasm. Forthe same reason, dehydrogenase reactions involving NAD(P)as electron acceptor might be expected in the cytoplasm(i.e., in the oxidation of formaldehyde, formate, or di- ortriethylamine).

Importance of reaction products to carbon assimilation(Table 3). In the CO-oxidizing or methylotrophic bacteria theproduction of CO2 from CO, HCHO, or HCOOH and theproduction of HCHO from CH30H, (CH3)3N, or (CH3)2NHare reactions critical to carbon assimilation as well as energyproduction. The achievement of an adequate concentrationof CO2 in the cytoplasm can be a significant growth-limitingproblem for photo- and chemoautotrophs. This idea is sup-ported by the presence of inducible carbonic anhydraseenzymes and inducible CO2 or bicarbonate uptake systemsin these organisms (24, 41, 78, 169). During growth onmethane, methanol, or methylated amines, a significantfraction of CO2 produced from the oxidation of formalde-hyde is incorporated into cellular carbon. Thus, generationof CO2 in the cytoplasm is desirable. An analogous situationis observed where growth is supported by the oxidation ofCO to CO2 by carboxydobacteria (85).Formaldehyde produced in the oxidation of methanol (15)

or tri- or dimethylamines is assimilated (12). It is possiblethat production of formaldehyde in the cytoplasm facilitatescarbon assimilation.

ACKNOWLEDGMENTS

We thank 0. H. Tuovinen, M. Beardmore-Gray, P. J. Chapman,T. C. Olson, S. E. Stevens, P. Mitchell, and M. E. Lidstrom for theirevaluation of this manuscript and useful suggestions. We also thankK. H. Nealson for providing unpublished information and usefuldiscussions. We are grateful to K. Backlund for typing the manu-script and K. Kohn for preparation of illustrations.This work was supported by grants from the National Science

Foundation (PCM 83-08766) and the U.S. Department of AgricultureCRGO (82-CRCR-1-1118).

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LITERATURE CITED1. Adair, F. W. 1966. Membrane-associated sulfur oxidation by

the autotroph Thiobacillus thiooxidans. J. Bacteriol.92:899-904.

2. Adams, M. W. W., L. E. Mortenson, and J.-S. Chen. 1981.Hydrogenase. Biochim. Biophys. Acta 594:105-176.

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