THE JOURNAL OF BIOLOGICAL CHEMISTRY ‘c’ 1984 hy The American Society of Biological Chemists, Inc.

Vol. 259, No. 1, Issue of January 10, pp. 124-133, 1984 Pr~nted in U.S.A.

Purification and Characterization of the Rieske Iron-Sulfur Protein from Thermus thermophilus EVIDENCE FOR A [2Fe-2S] CLUSTER HAVING NON-CYSTEINE LIGANDS*

(Received for publication, May 23, 1983)

James A. Fee$, Karen L. Findling, Tatsuro Yoshida, Russ Hille, George E. Tarr, David 0. Hearshen, W. R. Dunham, Edmund P. Days, Thomas A. Kent$, and Eckard Munck$B From the Biophysics Research Division and the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109 and §The Gray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392

We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for M, - 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mossbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mossbauer studies of the oxidized and reduced protein demon- strate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Moss- bauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for com- plete reduction of the protein and that the g = 1.94- type signal of the reduced protein has a spin concen- tration of one spin (S = 112) per 2Fe. Within the excellent resolution of the Mossbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues.

The Mossbauer spectra of the reduced protein were analyzed with an S = 112 spin Hamiltonian. The hyper- fine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fez+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [ZFe- 2S] center.

Proteolipid complexes containing cytochromes b and c are essential for respiratory electron transport in mitochondria ( I ) , chloroplasts (2,3), and certain bacteria (4,5). As isolated, these complexes contain two B-type hemes, one C-type heme, variable amounts of respiratory quinone, and a characteristic Fe/S protein. Based on analyses of total Fe and heme content of purified bc, complex from bovine tissue (6) and yeast cells (7), it seems likely that the iron-sulfur protein contains a

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

GM12176 and GM30974. .$ Recipient of United St.ates Public Health Service Grants

T Recipient of United States Public Health Service Grant GM22701.

[2Fe-2S] cluster. Indeed, Albracht and Subramanian (8) dem- onstrated the [2Fe-2S] characer of this protein by examining the EPR spectra of ”Fe-enriched membrane particles of Cun- dida utilis.

The Fe/S protein from the bcl complex was first purified from bovine mitochondria by Rieske et al. (9). Apparently for the lack of an appropriate terminology denoting its function in respiration, it is widely referred to as the Rieske iron-sulfur protein. The properties of this protein have recently been reviewed by Trumpover (10) and Malkin and Bearden (11): as isolated from the mitochondrial bc, complex, in a form free of detergent, the protein exhibits a M, = 24,500 upon SDS’ gel electrophoresis and contains approximately 1.4 g atom of Fe and 1.0 g atom of acid-labile sulfide (12). The Rieske protein isolated from bovine mitochondria by the method of Trumpower and Edwards (12) is quite unstable. However, it has been possible to reconstitute ubiquinol-cytochrome c re- ductase activity in bc, complex depleted of the iron-sulfur protein (12). Subsequent results have indicated that the Rieske protein acts as a ubiquinol-cytochrome c,/ubisemiqui- none-cytochrome 6 oxidoreductase within the bc, complex (13).

Previous work with Thermus thermophilus membranes (14) demonstrated the presence of a menaquinone-specific seg- ment of the respiratory chain capable of transferring electrons from NADH to partially purified Rieske protein. In this paper, we present a purification procedure for the Rieske protein from the membranes of T. thermophilus, describe several of its properties, and present evidence that each of the two [2Fe- 2S] clusters is coordinated to fewer than four cysteinyl resi- dues.

EXPERIMENTAL PROCEDURES

Materials and methods were generally as described previously (15). Enrichment of proteins with 57Fe was accomplished by growing the bacteria in a defined culture medium containing -5 pM 90% ”Fe. Thin layer isoelectric focusing gels (Servalyt Precote 3-10), running buffer, Serva Blue G stain, and isoelectric pH markers (test mixture 9) were purchased from Serva Fine Chemicals (Garden City Park, NY). The commercially available thin layer isoelectric focusing gels were soaked in a solution containing 1% laurylmaltoside, 10% sorbi- tol, and 3% Servalyt 3-10 carrier ampholytes prior to use and were run according to the manufacturer’s instructions with a LKB model

’ The abbreviations and trivial names used are: SDS, sodium dodecyl sulfate; laurylmaltoside, dodecyl-@-D-maltoside; octylgluco- side, n-octyl-fi-D-glucopyranoside; EDTA, ethylenediaminetetraa- cetic acid; Tris, tris(hydroxymethy1aminomethane; T E buffer, Tris. HCI at indicated concentration, 0.1 mM EDTA, pH = 7.8; TX-100, Triton X-100; PAGE, polyacrylamide gel electrophoresis; CD, circular dichroism; EFG, electric field gradient.

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Rieske Iron-Sulfur Protein from Thermus thermophilus 125

2117 flat bed electrophoresis apparatus a t 4 "C. Polyacrylamide gel electrophoresis in the presence of 8 M urea and SDS was carried out by the method of Swank and Munkres (16). Approximately 1 cm of stacking gel having half the total acrylamide concentration but iden- tical buffer composition as the main gel was used on top of the separating gel. A Hoeffer model SE500 slab gel electrophoresis system (Hoeffer Scientific, San Francisco, CA) was used with a 140 X 100 X 1.5 mm gel.

Optical spectra were recorded on either a Perkin-Elmer model 320 or a Cary model 219 double beam spectrophotometer. Anaerobic, optical reductive titrations were carried out in an all-glass device described elsewhere (17). CD and magnetic CD spectra were recorded on a Jasco model J-40C instrument a t room temperature. EPR spectra were recorded a t X-band using a Varian E-112 spectrometer and a t S-band using a home-built system with a quartz-filled dielectric cavity operating in the TE,,, mode (18, 19). Cryogenic temperatures were obtained with a helium flow system (20). Mossbauer spectrometry was carried out as described previously (21).

Protein was measured either by a modified procedure of Lowry et al. (22) using bovine serum albumin as the secondary standard and including 0.5% SDS in the alkaline copper solution (23) or by the modified biuret procedure of Janatova et al. (24). Iron analyses were done either by atomic absorption or according to the method of Massey (25), and sulfide was determined as described in Refs. 26 and 27. The initial dissolution of protein into 0.5 N NaOH required by the procedure did not result in denaturation. This is a dramatic demonstration of the protein's stability. Denaturation was accom- plished by heating the alkaline solution over a flame then quickly cooling to reduce the loss of H2S.

RESULTS

Purification Procedures-The Rieske iron-sulfur protein was initially demonstrated (14) by EPR analysis to co-frac- tionate with the NADH and succinic dehydrogenases during development of the first anion exchange column described in Ref. 15. Its purification is thus begun by combining all frac- tions from this column which exhibit NADH dehydrogenase activity. Unless otherwise noted, all steps were carried out at 4 "C. Starting with 2 kg of cell paste and carrying out the fractionation, the combined fractions have a volume of "3 liters. This was dialyzed with three changes against 16 liters of 75 mM Tris.HC1, 0.1 mM EDTA (TE buffer) and 0.1% TX-100 to remove salt. The solution was then brought to 2% TX-100, passed, a t room temperature, through a 5 X 25 cm column of DE52 equilibrated with the same buffer, and washed with 6 liters of 75 mM T E buffer, 2% TX-100. The column was developed with 75 mM T E buffer and a 0 to 0.3 M NaCl gradient of 2-liter total volume. The elution profile of 450 nm absorbing material is shown in Fig. 1A. The Rieske protein was located in this profile by its characteristic reduced minus oxidized difference spectrum. Those fractions contain- ing the iron-sulfur protein were combined (-500 ml), dialyzed against several changes of 10 mM TE buffer, 0.1% TX-100, and then passed over a 2.6 X 14 cm column of pre-equilibrated DE52. Protein was subsequently eluted from the column with 75 mM TE buffer, 0.1% TX-100, and 1 M NaCl in a volume of 50 ml. This solution was made 0.1% in octylglucoside and further concentrated to -15 ml using an Amicon stirred cell concentrator with a PM-10 membrane. This solution was loaded onto a Bio-Gel P-100 gel filtration column having a bed volume of 400 ml and equilibrated with 100 mM T E buffer in the absence of detergent. The column was developed with 100 mM T E buffer. Its elution profile is shown in Fig. 1B.

The first peak is yellow-colored and contains NADH de- hydrogenase activity while the second peak contains the pink- colored Rieske protein, as indicated by the difference optical spectrum (see Fig. 4 below). The two peaks were pooled separately and the yellow material frozen a t -10 "C for sub- sequent purification of the NADH dehydrogenase. The pink material (25 ml) was brought to 0.1% octylglucoside, concen- trated over a PM-10 membrane, diluted with a small amount

Anlon Exchange a

Anm Exchange/ C Gel Flltrotlon n

FRACTION NUMBER

FIG. 1. Purification of Rieske iron-sulfur protein from T. thermophilus. A , fractionation of starting material, obtained as described in Ref. 15, on DEAE-cellulose (DE52) with a salt gradient. Fractions containing the Rieske protien, (within the uertical bars) were concentrated and applied to a Bio-Gel P-100 gel filtration column. B, elution profile of the gel filtration column. The leading peak is yellow in color and contains NADH and succinic dehydrogen- ase activities. The trailing peak is pink colored and contains the Rieske protein. The fractions within the vertical bars were concen- trated and applied to a column of DEAE-Bio-Gel A-50 and eluted as shown in C with a salt gradient. Pure Rieske protein was obtained from fractions 78-86. Other details are given in the text.

of water, and concentrated to -2 ml having a conductivity of a 2 mmho.

The final stage of purification involves the use of combined anion exchange-gel filtration and is accomplished by one or two passes over a 1.5 X 20 cm column of DEAE-Bio-Gel A. The protein solution was applied to this column after equili- bration with 75 mM T E buffer and developed with a 180-ml 0 to 0.3 M NaCl gradient. A typical elution profile is shown in Fig. 1C. Material at this stage is nominally pure as indicated by gel electrophoresis and isoelectric focusing (Fig. 2) and has been used for preliminary characterization of the protein. Additional procedures which give effective purification in the latter stages are preparative level gel electrophoresis and isoelectric focusing. Typical yields of iron-sulfur protein are 0.3 to 0.6 pmol of protein.

The purification of the Rieske protein of Thermus is a straightforward extension of our previously reported purifi- cation of the cytochrome c,aa:, complex (15). The procedure and yields are both reproducible, and the Rieske protein behaves as a water-soluble protein, if a detergent is included during the concentration procedures to avoid an irreversible precipitation of the protein.

Evidence of Purity-Protein obtained by the above proce- dure was subjected to PAGE without prior denaturation. Only a single band was observed with Coomassie staining (not shown). SDS-PAGE of denatured protein showed the pres- ence of one band upon Coomassie staining (Fig. 2A) . Thin layer isoelectric focusing of the native protein also revealed the presence of one protein band at PI = 4.7 (Fig. 2B). Thus, the protein was found to be pure by three electrophoretic methods.

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126 Rieske Iron-Sulfur Protein from Thermus thermophilus

FIG. 2. Demonstration of the pu- rity and isoelectric point of the Rieske iron-sulfur protein isolated from T. thermophilus. A, SDS-poly- acrylamide gel electrophoresis in the presence of 8 M urea: total acrylamide concentration was 7% with a bisacrylam- ide to acrylamide ratio of 1:15. Samples were denatured in 4 M urea, 5% SDS, 4% P-mercaptoethanol, 10% glycerol, and 20 mM Tris.CI, pH 7.8 at 70 "c for 12 min. Gels were fixed and stained in 25% iso- propanol, 105 acetic acid, 0.2% Serva Blue G . RPand HHC indicate the Rieske protein and horse heart cytochrome c, respectively. Standards used were phos- phorylase b, bovine serum albumin, oval- bumin, carbonic anhydrase, soybean tr-ypsin inhibitor, and lysozyme. E , thin laver isoelectric focusing: for conditions, see "Experimental Procedures." Stand- ards and their isoelectric points on the /eft gel are: amyloglucosidase (3.5), con- albumin (5.9). horse myoglobin (7.3), whale myoglobin (8.3), and cytochrome c (10.7).

A

19.1 - RP

HHC -

kd - 92.5 I -66.2

pH 3.5 - - 45

B

8

Molecular Weight and Composition-Initial estimates of molecular weight were obtained from SDS-PAGE in gels of differing composition according to the method of Ferguson (28). These results are shown in Fig. 3 and suggest M , = 19,500 under the assumption that the protein is fully dena- tured.

Three independent iron analyses showed that the extinction coefficient a t 458 nm (the absorption maximum of the oxi- dized protein; see Fig. 4 below) was 3,000 f 100 [Fe]" cm". This was used in conjunction with protein analyses to further assess the composition of the protein. The Lowry method indicated 4,800 f 1,300 g of protein/g atom of Fe and the biuret method gave 6,600 f 1,400 g of protein/g atom of Fe. Determination of the amino acid composition gave the more precise figure for g of protein/g atom of Fe of 5,000 f 10%. Thus, there appears to be approximately 4FeIestimated M, - 20,000.

Parallel measurements of labile sulfide and Fe indicated 0.81 f 0.05 SZ-/Fe. Because of the inherent difficulties in this analysis and the rather harsh conditions required to denature the protein, we presume that this value represents a 1:l stoichiometry.

The amino acid composition was determined for three samples: apoprotein prepared by denaturation in 8 M guani- dine hydrochloride and 1 mM EDTA followed by dialysis, dried native protein extracted with 1 N HCl, and native protein. There were no significant differences between these samples. Cysteine was determined by prior oxidation to cys- teic acid with performic acid using a modification of standard methods (29). The amino acid composition shown in Table I is statistically significant a t a minimum M , = 20,300 (30). This is consistent with the value from SDS-PAGE and allows for the presence of 1 methionine/molecule. [2Fe-2S] clusters are generally thought to be associated with four cysteine residues. I t is therefore noteworthy that the ratio of cysteine to Fe is unity for the Rieske protein. Indeed, this result gave us the first clue that we were dealing with a unique [2Fe-2S] site.

Optical Spectra-The optical absorption spectra of the ox- idized (solid line) and reduced proteins (dashed line) are shown in Fig. 4. The oxidized protein has peaks at 325 and

- 3! C- 2 5

5.9 -

- 14.4

10.7- -

RP - pH 4.7 f .05

% TOTAL ACRYLAMIDE

0

% TOTAL ACRYLAMIDE

0 0 -

. 0 8 - 06 - +- 04 - 02

CK,

-02

- -

-

MOLECULAR WEIGHT ( k dolton) IO 20 30 40 50

CK,

O2 i: IO 20 30 40 50 MOLECULAR WEIGHT ( k dolton)

FIG. 3. Electrophoretic determination of the molecular weight of Rieske iron-sulfur protein from T. thermophilus according to the method of Ferguson (28). A, log of electropho- retic mobility relative to horse heart cytochrome c versus total acryl- amide concentration. The samples were denatured as described in Fig. 2. All gels contained 8 M urea with a 1:15 bisacrylamide: acrylamide ratio. 0, molecular weight standards (ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme); 0-X-X-x-0, Rieske protein. E , retardation coefficients (the slope of lines calcu- lated from A ) uersus molecular weight. RP indicates the Rieske protein at M, = 19,500.

458 nm and a distinct shoulder a t 560 nm. The reduced protein has maxima at 380,425, and 550 nm. The difference spectrum, reduced minus oxidized, is characterized by a strong negative feature near 470 nm. Extinction coefficients for these bands are given in Table 11. Highly purified protein has a purity index A460/A2Pa = 0.27.

CD spectra of the oxidized and the reduced protein are shown in Fig. 5. The oxidized protein (solid line) has a weak negative feature a t 675 nm, another negative band at 542 nm,

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Rieske Iron-Sulfur Protein from Thermus thermophilus 127

TABLE I Composition of Rieske FeIS protein from T. thermophilus

Residue No./20,300" Da

Asx 9 Glx 25

His 5 Ser 4

Thr 5 Pro 22 Ala 23 TY r 5 Val 23 Met 1 Ile 4 Leu 21 Phe 4 LY s 9 Cysb 4 Trp Fe 4 S2- 4

Arg 9

Gly 19

-

'Estimated from the minimization procedure of Hoy et al. (30). * On standard proteins, the error in the measurement of cysteine

ranges from 0.3 to 5% (G. E. Tarr, unpublished results).

011 I I I I I 300 400 500 600 700

WAVELENGTH inm)

FIG. 4. Visible optical absorption spectra of oxidized (-) and reduced (- - -) Rieske iron-sulfur protein from T. ther- mophilus. The extinction coefficient refers to the concentration of [2Fe-2S] clusters. The traces on the left are shown at half-intensity.

a rather complicated set of positive bands between 400 and 500 nm, and a dominant negative band at 375 nm. The reduced protein (dashed line) has a weak positive band at 660 nm, a narrow, negative band near 500 nm, at least two positive bands between 400 and 480 nm, and a strong negative band at 384 nm. The magnetic CD spectra of the oxidized protein (Fig. 5, solid line) shows a broad negative feature near 590 nm while that of the reduced protein (Fig. 5, dashed line) shows a broad negative feature around 530 nm. These spectra are quite different from those of other Fe/S proteins (cf. "Discussion").

Electron Stoichiometry-The oxidized protein was titrated with sodium dithionite under anaerobic conditions and ob- served to take up 0.53 electron/Fe (data not shown). A similar stoichiometry has been reported for adrenodoxin, putidare. doxin, and plant type ferredoxins (31) and suggests a close association between two Fe atoms in a single cluster.

EPR Spectra-As indicated in Fig. 6, the Rieske protein exhibits an EPR spectrum only after reduction. This signal can be observed at temperatures as high as 100 K without

TABLE I1 Molar absorbance and circular dichroism of Rieske protein from

T. thermophilus All t and At values are +"5%.

x 4[2Fe-2S] X Le (CD)" nm xcm" nm

Oxidized 325 11,500 314 +19.0 458 6,000 f 200 375 -18.1 560 (s) 3,000 542 -2.2

675 -1.8 Reduced 380 4,800 384 -18.7

425 3,800 450 +12.0 550 2,200 500 -7.9

660 +2.9 Oxidized minus 470 2,900'

reduced 572 2,0006 "Molar circular dichroism, based on the concentration of [2Fe-

2si. Difference absorption.

-

300 400 500 600 700 800

WAVELENGTH (nm) FIG. 5. Magnetic circular dichroism and circular dichroism

spectra of oxidized (-) and reduced (- - -) forms of the Rieske protein isolated from T. thermophilus. T, Tesla = 10,000 G. Intensities are referenced to the concentration of [2Fe-2S] clusters.

obvious line broadening. Neither oxidized nor reduced forms exhibit a significant EPR absorption a t g = 4.3, indicating the absence of an isolated high spin Fe"+ ion. The EPR spectrum of the reduced form is rhombic with principal g values of 2.02, 1.90, and 1.80. We have determined the spin concentration by comparing the doubly integrated spectrum, recorded at 30 K, with that of a standard Cu2'-EDTA sample. The result, 0.94 * 0.15 spin/2Fe, in conjunction with the iron determination suggests that the protein contains two [2Fe- 2S] clusters. We have also studied EPR spectra at S-band (3- GHz microwave frequency). At this frequency, the EPR lines are narrower than at X-band and distinct magnetic hyperfine broadening is observed when samples enriched in "Fe are studied. By using methods described previously (32), we have determined with Mossbauer spectroscopy that the protein was enriched in "Fe to 60 & 5%. With this information, we have simulated the S-band EPR spectra using the magnetic hyper- fine tensors obtained from the analysis of the Mossbauer spectra (see Table I11 below). The calculated spectra fit the data reasonably well (data not shown). The spectral pattern at g, = 2.02 for the [2"Fe-2S] component of the spectrum shows a pattern typical of [2Fe-2S] rather than [4Fe-4S] clusters (cf. Fig. 9 of Ref. 33).

Mossbauer Studies-A Mossbauer spectrum of the oxidized Rieske protein recorded at 4.2 K is shown in Fig. 7. The spectrum consists of a superposition of two quadrupole doub- lets of equal intensity. The solid line in Fig. 7 is the result of least squares fitting two pairs of lines with Lorentzian shape

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128 Rieske Iron-Sulfur Protein from Thermus thermophilus

1000 2000 3000

2 d2 ,

190 I80

I I I I I I I I I I 3100 3200 3300 3400 3503 3600 3700 3800 3900

MAGNETIC FIELD (Gauss)

FIG. 6. X-band EPR spectra of T. thermophilus Rieske iron- sulfur protein. Top: the upper spectrum is that of the oxidized protein recorded at a spectrometer gain of 1.25 X lo4, and the lower spectrum is that of the dithionite-reduced protein recorded a t a gain of 3.2 X 10'. The protein concentration was -1 mM. The conditions of recording were: microwave power, 10 milliwatts; microwave fre- quency, 9.210 GHz; modulation amplitude, 10 G at 100 kHz; scan rate, 1000 G/min; and T = 36 K. Bottom: expanded scale EPR spectrum of reduced Rieske protein. The concentration of protein was "0.5 mM protein. The conditions of recording were: microwave power, 1 milliwatt; microwave frequency, 9.208 GHz; modulation amplitude, 10 G a t 100 kHz; scan rate, 250 or 125 G/min; and T = 25

V I 1 1 I

-4 -2 0 2 4

VELOCITY ( m m l s e c )

FIG. 7. 4.2 K Mossbauer spectrum of '?Fe-enriched oxidized Rieske protein from T. thermophilus. The solid line is the result of least squares fitting two quadrupole doublets to the data. One doublet has A& = 0.91 mm/s and 6 = 0.32 mm/s; the other AEB = 0.52 mm/s and 6 = 0.24 mm/s. The absorption lines have a full width at half-maximum of 0.26 mm/s. Within the uncertainties, each of the four absorption lines contribute 25 rt 2% of the total absorption.

to the data. The four lines can be associated in two different ways, We have chosen that association (brackets in Fig. 7) which keeps the differences in the isomer shifts of both sites to a minimum. At 4.2 K, the Mossbauer parameters for the quadrupole splittings, A&, and the isomer shifts, 6, are AEf!(l) = 0.91 f 0.02 mm/s and 6(1) = 0.32 5 0.01 mm/s, and AEf,(2) = 0.52 +. 0.02 mm/s and 6(2 ) = 0.24 f 0.01 mm/s.

H = 4.0 T

Y 1 1 1 1 1 1 1 1 1 i

-4 -2 0 2 4

VELOCITY (mrnlsec)

FIG. 8. High temperature Mossbauer spectra of the reduced Rieske protein from T. thermophilus protein. A , zero-field spec- trum recorded a t 230 K. The solid line is the result of least squares fitting two doublets to the data. The lines of the ferrous doublet have 0.28 mm/s full widths whereas those of the ferric site have a full width of 0.35 mm/s. B, spectrum taken a t 200 K in a magnetic field of 4.0 T applied parallel to the observed y-radiation. The solid line is a theoretical spectrum computed with the parameters of Table 111. The dashed line is a spectrum of the ferrous site computed for V,.,, (Fe") > 0.

(The alternative association would yield isomer shifts of 0.18 mm/s and 0.38 mm/s which seems unreasonable to us.) The quadrupole splittings of both sites were found to be independ- ent of temperature in the range of 4.2 K < T < 195 K.

The observation of an EPR signal for the reduced Rieske protein shows that this state is paramagnetic. Unlike plant ferredoxins (33), the EPR signal can be observed even at temperatures above 100 K (cf. also Ref. 9). This shows that the electronic spin relaxation time is quite slow at 100 K. Fig. 8A displays a Mossbauer spectrum of the reduced ferredoxin recorded at 230 K. At this temperature, but not for T < 170 K, the spin relaxation rate is fast compared with the nuclear precession frequencies. The magnetic hyperfine interactions are, therefore, averaged out. The spectrum in Fig. 8A consists of two well resolved doublets with parameters typical of a high spin ferric and a high spin ferrous site. From the least squares fit displayed in Fig. 8A, we obtained: A&(Fei+) = 0.61 k 0.02 mm/s and 6(Fe"+) = 0.22 f 0.02 mm/s; &%,(Fez+) = 2.81 f 0.02 mm/s and 6(Fe2+) = 0.65 & 0.01 mm/s. As the temperature is lowered, the lines broaden appreciably due to the onset of magnetic hyperfine interactions. (Even at 230 K, the ferric doublet displays some relaxation broadening.) At temperatures below 160 K, the lines are too broad for an evaluation of the quadrupole splittings from the line positions. AEo(Fe2+) was found to be temperature-dependent with AE,(Fe'+) = 2.90 k 0.02 mm/s at 195 K. Within the uncer- tainties, AE,,(Fe:'+) is independent of temperatures as ex- pected for a high spin ferric site. Fig. 8B shows a spectrum taken a t 195 K in an applied magnetic field of 4.OT. As discussed below, analysis of this spectrum shows that the largest component of the EFG tensor of the Fez+ site is positive.

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Rieske Iron-Sulfur Protein from Thermus thermophilus 129

Fe"

1 , 1 # 1 1 1 1 -4 -2 0 2 4

VELOCITY (mmisec)

FIG. 9. 4.2 K Mossbauer spectra of the reduced Rieske pro- tein from T. thermophilus recorded in a parallel magnetic field of 60 milliTeslas. A , the solid line is a theoretical calculation using the parameters of Table 111. B, the solid line gives the theoretical spectrum for the ferric site, normalized to correspond to 50% of the total absorption. C, subtraction of the theoretical spectrum of B generates an "experimental" spectrum of the ferrous site (full circles). The solid line is a theoretical spectrum for the ferrous site.

Figs. 9A and 1OA show 4.2 K Mossbauer spectra of the reduced protein taken in magnetic fields of 60 milliTeslas applied parallel (Fig. 9A) and perpendicular (Fig. 1OA) to the observed y-radiation. From earlier work on other [2Fe-2S] proteins (34-36), the nature of these spectra is quite well understood and data analyses have been discussed in the literature. The Mossbauer spectra of the Rieske protein are strikingly similar to those reported for putidaredoxin from Pseudomonas putida (34) and, thus, many aspects of the data analysis described for the latter protein apply here as well. Considering that the protein contains 4 Fe atoms and about 4 S2-/molecule, it is worth noting that Mossbauer spectra observed here are drastically different from those observed for [4Fe-4S] clusters.

Analysis of Mossbauer Spectra-It has been shown (34-36) that the Mossbauer spectra of [2Fe-2S] clusters can be eval- uated by assuming that the observed spectra are superposi- tions of two equally intense component spectra, each describ- able by an S = I/z spin Hamiltonian of the form

where the electronic g tensor is common to both iron sites of the spin-coupled system. A ( i) is the magnetic hyperfine tensor of site i (i designates the Fez+ and the Fer'+ sites). The A tensors of Equation 1 can be related within the spin-coupling model of Gibson et al. (37) to the A tensors of the individual

l ' l ' l ~ l ' l

1 , 1 ~ 1 , 1

-4 -2 0 2 4

VELOCITY (mmisec )

FIG. 10. 4.2 K Mossbauer spectra of the reduced Rieske protein from T. thermophilus recorded in a magnetic field of 60 milliTeslas appliedperpendicular to the observed y-radia- tion. The figure is analogous to Fig. 9.

ions, ci(i), by ci(Fe:") = :%A(Fe,"+) and &(Fez+) = -3/iA(Fe2+). The EFG tensor of site i has principal axis components Vxx(i), V,,(i), and Vzz(i), with a(i) = (VJi) - V,,,(i))/Vzz(i) being the asymmetry parameter.

The solid lines in Figs. 9A and 1OA are the result of fitting the spectra of both iron sites to Equation 1 by means of computer simulations. The solid lines in Figs. 9B and 1OB represent the theoretical spectra of the ferric site. By subtract- ing the theoretical spectra of the latter from the experimental data, we obtained spectra for the Fez+ site (full circles in Figs. 9C and lOC). Finally, the solid lines in Figs. 9C and 1OC are theoretical spectra of the FeL+ site. The parameters used for the calculations are listed in Table 111. In the following, we discuss briefly the principal features of these spectra.

(a) The observed Mosshauer spectra reflect averages of randomly oriented molecules. Since the g tensor is fairly isotropic, the Mossbauer spectra do not provide information about the orientation of the A tensors and the EFG tensors relative to 2. For the same reason, the relative orientation of the hyperfine tensors of the ferric and the ferrous sites cannot be determined. However, electron nuclear double resonance studies of a variety of ferredoxins (36) have established such correlations. Inasmuch as the cluster of the Rieske protein yields Mossbauer spectra very similar to those of putidare- doxin (34) and adrenodoxin (35), it is reasonable to assume that the z axes chosen here coincide with g, = 2.02.

(b) The Fez'+ site has a fairly isotropic A tensor. The largest component of the EFG tensor, V,,(Fe:'+), is positive and the asymmetry parameter is v(Fe'") < 0.5. For 7 = 0, as chosen here, the spectra of the ferric site are little affected when A, and A, are interchanged.

(c) The A tensor of the Fez+ site is quite anisotropic. The

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130 Rieske Iron-Sulfur Protein from Thermus thermophilus TABLE 111

Mossbauer parameters for the reduced Rieske protein and Dutidaredoxin at 4.2 K

Rieske protein ~ PutidaredGG Mossbauer (Ref. 34) parameter

Ferric site Ferrous site Ferric Ferrous site site

8 (mm/s) 0.31 zk 0.03 0.74 f 0.03 0.35 0.65 AEQ (mm/s) 0.63 * 0.10 3.05" k 0.05 0.6 2.7" rl 0 0" 0.5 0" A , (MHz)b A, (MHz)

55 f 3 11 + 3 -56 +14

A , (MHz) 50 f 3 14 f 1.5 -50 +21 43 f 4 33 f 1.5 -43 +35

We have quoted AEQ and I) for the ferrous site in (x ' ,y ' ,z ' ) . This system has the z' axis along the x axis of the principal axis system (x,y,z) of theA tensor. In Ref. 34, AEQ(Fee+) and q(Fe2+) were quoted in (x,y,z). In that system AEQ < 0 and q = -3.

I, The coordinate system of the ferric and the ferrous sites do not necessarily coincide (see text).

EFG tensor of the ferrous site has axial symmetry around the x axis. In contrast, the A tensor is roughly axial about the z axis. The coordinate system (x',y',z') of the EFG tensor is commonly chosen such that 1 V,. , . I 2 I VY,.", I 2 I V,,,, I. In such a "proper" system, 0 5 t)' 5 1 and the sign of AE, is given by the sign of the largest component of the EFG tensor, V,.,,. For putidaredoxin (34), adrenodoxin (35), and the Rieske protein, the z' axis is along the x axis of the A tensor and V,',' > 0. In contrast, the ferredoxins isolated from plants (35) have V,,,, < 0 and the z' axis aligned with the z axis (corresponding to a ground state with d,' symmetry). We have made a direct determination of the sign of V,.,, by studying the reduced protein a t 200 K, in the limit of fast electronic spin relaxation, in an external field of 4.0T. The shape of such spectra is fairly independent of the A values (see Lang (38) and Munck and Zimmermann (39)). The solid line in Fig. 8B was computed with the parameters of Table 111, assuming fast electronic relaxation. The dashed line shows the spectrum of the ferrous site, computed for V,,,, > 0. For V2,zf < 0 the theoretical spectrum would reverse left to right and the triplet pattern would appear at the high energy side of the spectrum; V,.,, < 0 is clearly incompatible with the data.

After we obtained a satisfactory parameter set, we coupled our simulation program to a least squares fitting routine. This allowed us to explore more systematically whether there are other regions in parameter space where satisfactory fits could be obtained. In particular, we investigated whether the EFG tensor of the ferrous site can be rotated relative to the prin- cipal axis frame of the A tensor. In all instances, the least squares fitting routine converged back to the solution of aligned tensors, i.e. the symmetry of the Fe2+ site is not lower than rhombic. We also found no satisfactory solution for I)'(Fe2+) > 0.2, i.e. the field gradient is axially symmetric.

DISCUSSION

Our major conclusions are that the Rieske protein of T. thermophilus contains two [2Fe-2S] clusters and that each of these has at least two non-cysteine ligands. We will consider the evidence for these conclusions after first discussing our other results.

The Rieske protein of T. thermophilus is very similar to that of mitochondrial and bacterial systems. Thus, the optical spectrum of the Thermus Rieske protein is qualitatively in- distinguishable from those of mitochondrial protein purified from beef heart (9, 40) and Neurospora (41). Likewise, the EPR spectrum is identical with that observed with purified bcl complex (7) and intact bacterial membranes (11, 42). The

molecular weight of the Thermus Rieske protein, M , - 20,000, is slightly smaller than that reported for the beef (12) and Neurospora (41) proteins. However, there may be more im- portant differences between the Rieske protein from Thermus and that from mitochondrial sources. The presence of two [2Fe-2S] clusters in the Thermus protein was not anticipated from iron analyses of purified mitochondrial bcl complex (6, 71, which suggested that only one [2Fe-2S] cluster could be associated with the Fe/S protein (see also Refs. 41 and 43). It is thus possible that the proteins from these sources have intrinsically different iron contents or that significant amounts of Fe and/or Rieske protein are lost during purifi- cation of the bcl complex.

Optical and Dichroic Spectral Properties-The spectral properties of the Rieske protein are decidedly different from other [2Fe-2S] ferredoxins (44, 45). Thus, the distinct maxi- mum in the optical absorption spectrum is red-shifted some 40 nm in comparison to plant ferredoxins and putidaredoxin (44), and the molar absorbance index of the cluster is approx- imately two-thirds as large as that of the above-mentioned [2Fe-2S] proteins. The CD spectra of oxidized and reduced Rieske protein are distinctly different from those previously observed for [2Fe-2S] centers (45). Notably, the intense band near 420 nm, thus far characteristic of [2Fe-2S] proteins, is absent. These spectra suggest that the molecular asymmetry experienced by the [2Fe-2S] clusters in the Rieske protein is quite different from that of other [2Fe-2S] proteins.

Mossbauer Spectra-The Mossbauer spectra of Figs. 9 and 10 are strikingly similar to those reported for putidaredoxin from Pseudomonas putida (34), an electron carrier (E, = -235 mV (46)) and effector in the cytochrome P-450 camphor hydroxylation system. As can be seen from an inspection of Table 111, the Mossbauer parameters of the Fe3+ site are the same for both proteins. However, there are differences at the Fe'+ site. The y components of the A tensor are distinctly smaller for the Rieske protein, while A, and A, are about the same for both proteins. The EFG tensor of the Fez+ site of both proteins is axially symmetric around the x axis of the A tensor. Moreover, the largest component of the EFG tensor is positive for both proteins (see Footnote a of Table 111). In contrast, the [2Fe-2S] ferredoxins derived from plants (35) have an EFG tensor which is axially symmetric around the z axis, the largest component being negative. Such a field gra- dient is expected for an Fe2+ ion with an orbital ground state having dZ2 symmetry. The EFG tensor observed for the Rieske protein could possibly reflect an orbital with dx2 - p symmetry. An alternative explanation has been given by Bertrand and Gayda (47). According to their model, the Fez+ sites of the various proteins experience rhombic distortions of different strength. By allowing one orbital of the t22 set to mix into the cl,' ground state, Bertrand and Gayda have rationalized the observation of a positive V2,2 , for putidaredoxin. We should point out, however, that the model predicts t)' = 0.75 for putidaredoxin which is too large compared to the experimen- tal value of 7' = 0. Interestingly, the Rieske protein has t)' = 0 as well.

Evidence for Non-cysteine Ligands-Two aspects of our results are especially noteworthy. First, chemical analyses have revealed that the protein contains iron, sulfide, and cysteine in equal molar ratios, four of each per molecule. Second, the Mossbauer data prove that the Rieske protein contains [2Fe-2S] clusters. The spectra are totally unlike those observed for [4Fe-4S] clusters; in particular, [4Fe-4Sl clusters do not have a site with a localized Fez+-valence as observed here. The iron anslyses and the Mossbauer data, taken together, imply that the protein has two [2Fe-2Sl clusters. This conclusion is further supported by the obser-

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Rieske Iron-Sulfur Protein from Thermus thermophilus 131

Y- -CH2-S-

\ A /y-

/"\ /Fe\ -C%-S" S= X- -X S= -S-CH2-

A B SCHEME 1

vations that two electrons are required for full reduction, that the Mossbauer data show each cluster accepts only one elec- tron, and that spin quantitations of the reduced protein reveal 1 spin/2Fe. In addition, the Mossbauer and EPR spectra, both sensitive to structural details, show that the two clusters are indistinguishable. The data thus imply that each cluster is coordinated to less than four cysteine ligands. Further, since it is unlikely that each Fe is coordinated to only three atoms, namely two S'- and one cysteine sulfhydryl, there are proba- bly ligands other than sulfur coordinated to the Fe. Possible liganding groups are imidazole, phenolate,' hydroxyl, and carboxylate. The following scheme suggests two plausible structural models (Scheme 1) for the [2Fe-2S] binding sites in Rieske protein (no particular geometrical arrangements are implied).

Redox Potential-The redox potentials of a variety of mi- tochondrial and bacterial Rieske proteins have been measured in situ. The E, values were observed in the range from +150 to +330 mV (10, 11).3 These values are some 400-600 mV higher than those observed for other [2Fe-2S] ferredoxins. Although the overall potential is determined by a variety of complex factors, the ligands to the Fe atoms are expected to play a major role. Johnson and Holm (50) have shown for [4Fe-4S] clusters that replacement of thiolate by carboxylate ligands results in substantial positive shifts of the redox potential ( - 100 mV/substituted ligand). Thus, we can expect that coordination of residues such as glutamate or aspartate would raise the potential of a [2Fe-2S] cluster. However, recent studies by Cleland et al. (51) have demonstrated that a shift to lower potentials occurs upon substitution of thiolate by phenolate ligands. At this point, of course, it is impossible to rule out other means of redox potential adjustment, such as substitutions of thiolate by nitrogen-containing residues or an increase in coordination number.

EPR Spectra-The g values observed for reduced [2Fe-2S] clusters have been successfully explained by the spin-coupling model of Gibson et al. (37). In this model, the deviations from g = 2 of the two high field resonances, generally labeled as g, and g?, are attributed primarily to the ferrous site, A large body of data from [2Fe-2S] ferredoxins has shown that most of the binuclear clusters have g,, = (g, + gy)/2 1.94. For these proteins, Bertrand and Gayda (47) have developed a ligand field model which focuses mainly on a rhombically distorted Fe2+ site. Restricting their analysis to clusters with g,, 1.94, these authors have explained the pattern of g value variations among the ferredoxins quite well. On the other hand, the Rieske protein, excluded from their analysis, has g,,,, = 1.85. Unusual g values such as those observed for the Rieske protein caught the attention of Blumberg and Peisach (52) who, in 1974, analyzed the g values of 27 ferredoxins.

~~ ~

Preliminary resonance Raman studies have failed to detect Ra- man scattering lines characteristic of tyrosine (48) under conditions which readily express the Fe-S stretching modes of the cluster (49) (K. Shaw and J. A. Fee, unpublished observations).

The Thermus protein is readily reducible by ascorbate, suggesting that the (2Fe-2SI centers have reduction potentials well above 0 V (NHE).

Among these were two proteins, namely 4-methoxybenzoate- 0-demethylase and coenzyme Q-cytochrome c reductase, hav- ing a low g,, value just like the Rieske protein. From a comparison of the g values within the body of data including that from clusters for which selenium was substituted for labile sulfur, Blumberg and Peisach concluded, that "the chemical make-up of the paramagnetic site of these two enzymes must include one or more atoms which are less electron donating than sulfur."

Disposition of the Non-cysteine Ligands-X-ray diffraction studies of the [2Fe-2S] ferredoxin from Spirulina platensis have revealed that four cysteine residues are coordinated to the cluster (53). This coordination had been expected from prior analyses of a large body of physicochemical data (see for instance Refs. 36 and 44). Moreover, the spectral proper- ties of the synthetic analogues (54) developed in R. H. Holm's laboratory give strong support for this coordination. Indeed, the combined structural and spectral data may lead one to presume that all [2Fe-2S] clusters are attached to the protein matrix by four cysteine ligands. However, the differences among individual [BFe-2S]-containing proteins, e.g. plant fer- redoxins and putidaredoxin, are often distinct, and one cannot exclude the possibility that some proteins may utilize one or two non-sulfur ligands. Our data suggest that the Rieske protein may use two ligands other than cysteine for the cluster coordination. If we assume that there are indeed two such ligands (X and Y , including the possibility X = Y ) , it would be interesting to know whether both non-cysteine ligands are coordinated to the same iron as depicted in Scheme lA, or whether X and Y are coordinated to different irons as indi- cated in Scheme 1B.

In order to address this question, we consider the isomer shifts obtained from Mossbauer studies. Isomer shifts are good markers for the oxidation states of high spin compounds, and the tetrahedral environment of sulfur ligands yields shifts which are in the lower ranges of the values observed for high spin ferric and ferrous compounds. In proteins, the range of 6 values is 0.25 mm/s < 6 < 0.5 mm/s for high spin Fez+ and 0.70 mm/s < 6 < 1.3 mm/s for high spin Fe2+ at 4.2 K. For example, the tetrahedral sulfur environment of the single iron of rubredoxin (55) produces 6(Fe3+) = 0.32 mm/s and 6(Fe2+) = 0.70 mm/s. Inasmuch as a tetrahedral sulfur environment yields the smallest observed shifts, one expects an increase in 6 if an oxygen or nitrogen ligand is substituted for a sulfur ligand. To date no [2Fe-2S] model complexes are available for which thiolate ligands have been substituted by oxygen- or nitrogen-containing functional groups. Some information, however, is available from Mossbauer studies (56) of [S,M~S,Fe(S-phenol),]~-. The core structure (56) of this di- mer, [MoFe-ZS], is very similar to that of [2Fe-2S] clusters. Mossbauer and susceptibility studies have shown (56) that the electronic spin of the cluster is S = 2 and that this spin originated from a high spin ferrous ion. An isomer shift 6 = 0.47 mm/s was observed at 4.2 K. Silvis and Averill (57) have synthesized an analogous compound in which the two thio- phenolate ligands coordinated to the iron are replaced by phenolate. The phenolate-substituted complex exhibits 6 = 0.57 mm/s. Although the absolute values of 6 are somewhat shifted relative to those of the Fe'+ sites of reduced [2Fe-2S] clusters, reflecting the presence of molybdenum, the increase in 6 of 0.1 mm/s upon substitution of sulfur by oxygen is in the expected direction.

In Table IV, we have listed the isomer shifts of the Fe:'+ and Fe2+ sites for a variety of ferredoxinsP Most of these

I t would be interesting to compare these shifts with those of model complexes known to have sulfur ligands coordinated to the

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132 Rieske Iron-Sulfur Protein from Thermus thermophilus

TABLE IV Isomer shifts of the Fe3+ and Fez+ sites of reduced [2Fe-2S] clusters

Shifts are quoted at 200 K relative to metallic iron at room temperature. The uncertainties of the individual shifts are f0.02 mm/ s, not including the uncertainties introduced by applying second order Doppler shifts to the 4.2 K putidarcdoxin data and to the 150 K 4- methoxy-0-demethylase data.

Organism (protein) (Ref.) 6(Fe3') a(Fe2')

Spinach (ferredoxin) (58) 0.22 0.59 Scenedesmus (ferredoxin) (58) 0.22 0.59 Synechococcus liuidus" (ferredoxin) 0.25 0.57 Halobacterium (ferredoxin) (59) 0.30 0.55 P. putidab (putidaredoxin) 0.29 0.59

7'. thermophilus (Rieske protein)' 0.25 0.68 P. putida (4-metho~y-O-demethylase)~ 0.23 0.68 P. putida (benzene dioxygenase) (61) 0.25 0.68

" W. R. Dunham, unpublished results; quoted values are averages of results obtained at 175 and 225 K.

'Obtained from the 4.2 K values, 6(FeR+) = 0.35 mm/s and 6(Fe'+) = 0.65 mm/s. The values listed above have been shifted by -0.06 mm/s to account for the second order Doppler shift.

' This work. Obtained from 150 K data of Bill et al. (60) by applying a of -0.02

mm/s second order Doppler shift.

shifts were determined at temperatures near 200 K, where the electronic spin relaxation time is sufficiently fast for the observation of resolved quadrupole doublets. The relaxation rate of putidaredoxin is slow at 200 K (34); therefore, we have used the shifts obtained at 4.2 K?

Inspection of the 6(Fe") column in Table IV reveals that most [2Fe-2S] proteins examined have 6 values in the range 0.55-0.59 mm/s. In contrast, the ferrous site of the Rieske protein, but not the ferric site, has a distinctly more positive shift (6(Fe") = 0.68 mm/s) than these proteins. (The possible correlation of 6(Fe'+) with the redox potential of the cluster is noteworthy.) These data and the information obtained for the [MoFe-BS] dimer suggest that the non-cysteine ligands are coordinated to the ferrous site. If the two non-cysteine ligands of the Rieske protein are alike, we expect both to belong to the same iron; otherwise we would expect the shifts of both sites to have increased. Moreover, if the isomer shifts indeed reveal the site containing the non-sulfur ligands, one would expect that this is also borne out in the shifts observed for the oxidized cluster. This appears to be the case. The oxidized Rieske protein exhibits two sites with distinctly different shifts, 6(1) = 0.32 and 6(2) = 0.24 mm/s, suggesting that it is site 1 which has the non-cysteine ligands and which

[2Fe-2S] core. Mascharak et al. (62) have reported Mossbauer spectra believed to belong to the trianion [Fe2S2((SCH2)2CsH4-o)2]3-. Beard- wood et al. (63) have reported different spectra and have criticized the interpretation of Mascharak et al., concluding that the Mossbauer spectra of the Mascharak et al. do not belong to the EPR-active trianion. W e share this concern. The 4.2 K spectra of Beardwood et al. have clearly the features of reduced [2Fe-2S] clusters. The value 6(Fe2+) = 0.70 mm/s a t 4.2 K corresponds to 6(FeZ') = 0.64 mm/s at 200 K, falling between the two groups of proteins listed in Table IV. However, inspection of the spectra and the spectral simulations (63) suggests that the accuracy of 6(Fe2+) is f 0.1 mm/s at best. Thus, the model complex data are not of much help for our present discussion. ' The measured isomer shift contains two contributions: a chemical

shift depending on the s electron density of the '7Fe nucleus and a second order Doppler shift, the latter being related to the specific heat (see Ref. 38). If no major structural changes occur between 4.2 and 200 K, and there is no evidence for this in putidaredoxin, then only the second order Doppler shift is temperature dependent. We have found in our laboratory that the 6 values of proteins typically shift by about 0.06 mm/s to lower energy when the temperature is raised from 4.2 to 200 K. For the Rieske protein, the shifts are a(4.2 K) - 6(195 K) = 0.055 mm/s for both the oxidized and the reduced state.

TABLE V Comparison of the physical properties of some NADH-dependent, [2Fe-2S]-containing aromatic oxygenases with those of Thermus

Rieske protein

Organism (activity) (Ref.)

P. putida (benzene dioxygenase) (64)

(4-Methoxybenzoate- 0-demethylase) (65)"

(67) (Toluene dioxygenase)

P. arvilla (benzoate- 1,2-dioxygenase) (68, and references therein)

Pyrazon-degrading bacteria (pyrazon dioxygenase) (69)

Thermus Rieske pro- teinb

Molecular A-= weight

[ZFe-ZSl) (number Oxidized Reduced

EPR g values

nrn 215,000 -340 410 (s)

(2) -460 -530 -560 (s)

120,000 410 375 2.008 (66) (2) 455 412 1.913

570 (ws) 518 1.72 150,000 326

(2) 450 550 (s)

200,000 325 375 (3) 464 -520

560 (s)

180,000 445 -375 2.02 (1) 545 (s) -420 1.91

520 1.79 20,000 325 380 2.02

(2) 458 425 1.90 560 (s) 512 1.80

May not be a dioxygenase. This work.

accepts the electron upon reduction. If, however, X and Y were very dissimilar ligands, we cannot rule out that they are coordinated to different irons; the lack of suitable model complexes does not allow us to make firm predictions about differential isomer shift changes.

Analogy to NADH-dependent Dioxygenases-While the properties of the [2Fe-2S] clusters of the Thermus Rieske protein are distinct from ferredoxins, we note strong similarities6 with the NADH-dependent, iron-sulfur-contain- ing dioxygenases found in various microorganisms. The com- ponent designated A1 (64) of these systems contains [2Fe-2S] clusters having properties remarkably similar to those re- ported here for the Thermus Rieske protein. In Table v, we have summarized the molecular weights, cluster composition, optical absorption, and EPR spectral properties of dioxygen- ases having a variety of specificities. In Table IV, we also listed the isomer shifts of the reduced [ZFe-ZS] clusters of two NADH-dependent oxygenases. Perusal of this informa- t,ion will reveal the remarkable similarity of the [2Fe-2S] clusters in these enzymes with those of the Rieske protein. It is thus likely that all these proteins contain the same type of [2Fe-2S] center. We suspect that these enzymes, which ap- parently have a Fe" ion at the active site, in addition to the [2Fe-2S] cluster(s), utilize a Rieske type center because of its intrinsically higher redox potential.

Achnouledgments-We thank Keith Shaw for recording resonance Raman spectra and Dr. S. Krimm for the use of his equipment. Dr. M. G. Choc assisted in the early stages of the work.

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P Day, T A Kent and E MünckJ A Fee, K L Findling, T Yoshida, R Hille, G E Tarr, D O Hearshen, W R Dunham, E

thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands.Purification and characterization of the Rieske iron-sulfur protein from Thermus

1984, 259:124-133.J. Biol. Chem. 

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