bacterial iron-sulfur proteins · the sulfur atoms in the prosthetic group recog-nizes that the...

Vol. 43, No. 3MICROBIOLOGICAL REVIEWS, Sept. 1979, p. 384-4210146-0749/79/03-0384/38$02.00/00

Bacterial Iron-Sulfur ProteinsDUANE C. YOCH'* AND ROBERT P. CARITHERS2

Department of Biology, University of South Carolina, Columbia, South Carolina 29208,' and Department ofBiochemistry, Rice University, Houston, Texas 770012

INTRODUCTION 384FERREDOXINS 385

Properties of Fe2S2* Ferredoxins 385Properties of Fe4S4* Ferredoxins 386

IRON-SULFUR ENZYMES 391Iron-Sulfur Enzymes 391Hydrogenase 391w-Hydroxylase 3934-Methoxybenzoate-O-demethylase 393Iron-sulfur proteins bound to procaryotic photosynthetic membranes 394Glutamine phosphoribosyl pyrophosphate amido transferase 396

Iron-Sulfur-Thiamine Pyrophosphate Enzymes 396Pyruvate dehydrogenase 396

Iron-Sulfur-Flavin Enzymes 397Succinate dehydrogenase 397Reduced nicotinamide adenine dinucleotide dehydrogenase 398Pseudomonas formate dehydrogenase 398Dihydroorotate dehydrogenase 399Glutamate synthase 399Adenylyl sulfate reductase 399Trimethylamine dehydrogenase 400

Iron-Sulfur-Heme Enzymes 400Sulfite reductase (dissimilatory) 400

Iron-Sulfur-Molybdenum Enzymes 400Nitrogenase 400Nitrate reductase (dissimilatory) 402C02 reductase (formate dehydrogenase) 403Iron-sulfur-molybdoprotein of unknown function 403

Iron-Sulfur Enzymes with Two or More Additional Cofactors 403Sulfite reductase (assimilatory) 403Coliform formate dehydrogenase 404Xanthine dehydrogenase 404

FERREDOXIN-DEPENDENT REACTIONS 405Clostridia 405Desulfovibrio 406Photosynthetic Bacteria .. 407Aerobic Bacteria .. 408Others ... 409

CONCLUDING REMARKS 409LITERATURE CITED 410

INTRODUCTION

Iron-sulfur proteins constitute a group of elec-tron transport proteins (ferredoxins) and oxida-tion-reduction enzymes whose function is nowknown to be central to such important cellularprocesses as photosynthesis, nitrogen fixation,respiration, and carbon dioxide fixation. Therelationship between the ferredoxin electron car-riers and iron-sulfur-containing oxidoreductasesmay be thought of as analogous to that betweencytochromes and heme-containing enzymes, re-spectively. The ferredoxins differ from their cy-

tochrome counterparts in that they generallyfunction at negative oxidation-reduction poten-tials (-340 to -480 mV), although in recentyears iron-sulfur centers in some enzymes havebeen determined to be as positive as +50 mV,and the high-potential iron protein (HiPIP), aferredoxin of unknown function, has a redoxpotential of approximately +350 mV.The distinguishing feature of iron-sulfur pro-

teins is the prosthetic group, which containsiron, commonly referred to as nonheme iron(16), bound to the peptide chain through fourcysteinyl-sulfur ligands. The simplest group of

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BACTERIAL IRON-SULFUR PROTEINS 385

nonheme iron proteins, the rubredoxins, containa single iron atom bound to the peptide by fourcysteinyl-sulfur bonds. Because rubredoxins lackinorganic sulfur, a characteristic common to thevast majority of iron-sulfur proteins, they arenot discussed here (see Lovenberg [162] for areview of this topic). The classical nonheme ironproteins contain inorganic or "acid-labile" sulfur(designated S*) in addition to the nonheme ironatoms. The term acid-labile as a description forthe sulfur atoms in the prosthetic group recog-nizes that the sulfur has a -2 valence and thatacid treatment liberates it as H2S during dena-turation of the protein. The current conceptconcerning the iron-sulfur protein prostheticgroup is that it is limited to clusters of eithertwo irons and two sulfides (Fe2S2*) or clusters offour irons and four sulfides (Fe4S4*) (Fig. 1). Anyindividual protein would therefore contain oneor more copies of these basic Fe-S structures. Ina number of iron-sulfur enzymes, one or more ofthese clusters function in conjunction withheme, flavin, pteridine, or molybdenum cofac-tors or with a combination of these cofactors. Inthe molybdenum-containing enzymes there isthe possibility that an iron-sulfur-molybdenumcenter may also exist and play an important rolein nitrogenase (215), whereas the evidence forsimilar centers in xanthine oxidase and certainnitrate reductases is less convincing.

In the 15 years since Bacteriological Reviewspublished what appears to be the first reviewarticle on iron-sulfur proteins (290), over 100iron-sulfur proteins have been isolated from var-ious plant, mammalian, and bacterial sourcesand studied in varying detail, resulting in over1,000 research publications. Because of thisenormous literature, this review focuses only onthe bacterial iron-sulfur proteins, and further-more, it is restricted to the more recent devel-

Acys- 5/ s -cys

Fe Fe

5"s-g\s / Fc\s-cys

B

CYS-S--Fe ss-cys

Fe

CY$S--Fe SFIG. 1. (A) Proposed model ofan Fe2S2* cluster of

ferredoxin. (B) Model of an Fe4S4* cluster of a bac-terial ferredoxin derived from X-ray diffraction stud-ies.

opments in this field. It is our goal to discuss thephysical and chemical nature of the bacterialiron-sulfur clusters as they are found in enzymesand ferredoxins and to relate this information tothe general physiology of the cell. More detailedinformation on individual iron-sulfur proteinsand the function of these proteins, includingthose from plant and animal sources, may befound in recent books edited by Lovenberg (163),the definitive source, and Neilands (194) and inreview articles (170, 223).

FERREDOXINSProperties of Fe2S2* Ferredoxins

Although it was thought for some time thatthe 2Fe-2S* ferredoxins were restricted to plantchloroplasts (266) and blue-green algae (cyano-bacteria) (249) (they are still referred to as"plant-type" ferredoxins), they have also beenfound in bacteria (Table 1); however, they arerelatively rare compared to those with tetranu-clear iron clusters (see Table 2).The structure of the Fe2S2* cluster (Fig. 1A)

was originally derived indirectly from a largebody of magnetic and spectroscopic data (39, 77,283), and a recent X-ray crystallographic analy-sis of a two-iron ferredoxin from Spirulina pla-tensis has confirmed this structure (287). Theiron atoms of the Fe2S2* cluster are bound to thepolypeptide chain by four cysteinyl-sulfur resi-dues and to each other by two bridging labilesulfur ligands. The iron atoms are nearly tetra-hedral in their coordination to the four sulfuratoms.

Iron-sulfur compounds of the general formu-lation [Fe2S2 (SR)4]2- have been synthesized andcrystallized, and X-ray analyses of these crystalshave shown their structure to be nearly identicalto that of the 2Fe-2S cluster shown in Fig. 1(173). Furthermore, the observation that theFe2S2* center from the ferredoxin of Spirulinamaxima (a cyanobacterium) can undergo thiol-ate substitution reactions to forn a product in-distinguishable from the synthetic analog (219)provides additional evidence that the Fe2S2*centers of the two-iron ferredoxin and the syn-thesized cluster are identical. In the extrusionreaction (121) thiolate groups, RS-, substitutefor the cysteinyl sulfur which binds the Fe-Sgroup to the peptide, allowing the iron-sulfurcore (either Fe2S2* or Fe4S4*) to be removedintact from the peptide. This reaction is shownby the equation:[Fe2S2*(SR)4]2- + 4R'SH

[Fe2S%*(SR')4]2 + 4RSHwhere R'SH represents thiol.

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TABLE 1. Properties of bacterial Fe2S2* ferredoxinsRedox EPR propertiespoten-

Compound Mol wt tial Symme- Biological function Reference(s)(E.) g values try(mV)

Pseudomonasputida ferredoxin 12,000 -235 1.94, 2.01 Axial Camphor hydroxylation 63, 282Halobacterium halobium ferre- 14,800 -345 1.90, 1.97, 2.07 Rhombic a-Keto acid oxidation 142

doxinEscherichia coli ferredoxin 12,600 -380 1.94, 2.02 Axial Unknown 147, 296Agrobacterium tumefaciens ferre- ND' -223 1.94, 2.02 Axial Unknown 293

doxinClostridium pasteurianum para- 25,000 -300 1.94, 1.96, 2.0 Rhombic Pyruvate oxidation 52, 115magnetic protein

Azotobacter vinelandii Fe-S pro- 21,000 -350 1.92, 1.94, 2.02 Rhombic Unknown 72, 157, 240tein Ib

Azotobacter vinelandii Fe-S pro- 21,000 -225 1.91, 1.93, 2.03 Rhombic Protection of nitrogenase 21, 157, 233,tein II from 02 240

Nostoc strain MAC ferredoxin I 12,050 -350 1.92, 1.96,2.06 Rhombic Photosynthesis, N2 and 49, 127, 128NGO- reduction

Nostoc strain MAC ferredoxin II 12,250 -455 ND ND ND 49, 127, 128

a ND, Not determined.bNot to be confused with A. vinelandii ferredoxin I (Table 2).

Electron paramagnetic resonance (EPR) spec-troscopy, which reveals paramagnetic centers,shows that the reduced Fe2S2* center has aprominent signal at g = 1.94, as shown in Fig. 2by the two-iron center of the spinach ferredoxin.(An exception to this is the Rieske protein[225], which is discussed in detail below.) Evi-dence suggests that the Fe2S2* cluster of a fer-redoxin has only two oxidation states. Moss-bauer spectroscopy data indicate that both ironatoms are in the ferric state in the oxidizedcluster and that, on reduction of the cluster bya single electron, one of these iron atoms isreduced to the ferrous state (77, 102) and theiron-sulfur cluster becomes paramagnetic due tothe unpaired electron on the ferric atom. TheEPR data, along with the data derived fromstudies of the 2Fe-2S analogs, indicate that theFe2S2* cluster in both ferredoxins and iron-sulfurenzymes functions between the (Fe2S2*S4CYS)2-and (Fe2S2*S4c"S)I valence states. The midpointpotential (Em) of this transition varies from oneferredoxin to another, but it is in the range of-250 to -400 mV (Table 1); in iron-sulfur en-zymes the Em's of the Fe2S2* cluster appear tobe more positive. In addition to the changesnoted in the magnetic properties of the Fe2S2*cluster, chemical reduction of this center is alsoaccompanied by a decrease of about 50% in thevisible absorbance bands located at approxi-mately 330, 420, and 460 nm.

Reconstitution of a typical 2Fe-2S* protein(Pseudomonas putida ferredoxin) with eithers7Fe or 3S resulted in line broadening of theEPR signal and provided strong evidence for theinvolvement of both the iron and the sulfide inthe electron-accepting center, a similar obser-

vation was made when either the labile sulfur orcvsteinyl sulfur was chemically exchanged with*1S (282; unpublished data cited in reference199). The strict conservation of the cysteinepositions in the amino acid sequence of theFe2S2* ferredoxins from plants and cyanobac-teria further suggests involvement of cysteinylsulfur in the active center (113, 309).The molecular weights of the two-iron ferre-

doxins from plants and cyanobacteria are, with-out exception, about 12,000, but the molecularweights ofthe two-iron ferredoxins from bacteriafall into two groups, one 12,000 and the otherapproximately 24,000. The significance of thisdifference in molecular weights is not yet under-stood, but it may in some way be related to thebiochemical function of these proteins. The12,000-dalton ferredoxins from P. putida andHalobacterium halobium play conventionalelectron transport roles in such reactions as cam-phor hydroxylation (136) and a-keto acid oxi-dation (141), whereas one of the 24,000-daltonFe2S2* proteins, Azotobacter iron-sulfur proteinII (72), is reported to play a role in protectingthe nitrogenase from 02 inactivation (233). Thefunction of the other two-iron ferredoxins, suchas those from Escherichia coli (296), Clostrid-ium pasteurianum (115), and Agrobacteriumtumefaciens (293), is unknown.

Properties of Fe4S4* FerredoxinsThe structure of the Fe4S4* cluster has been

determined by X-ray crystallographic studies oftwo bacterial iron-sulfur proteins, the 4Fe-4S*HiPIP from Chromatium vinosum (54) and the8Fe-8S* ferredoxin from Peptococcus aerogenes(246) (the latter of which contains two Fe4S4*

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clusters). The Fe4S4* centers from these twobacterial electron carrier proteins were found tobe nearly identical (Fig. 1B). As Fig. 1B shows,the Fe4S4* cluster has sulfide bridges betweeneach of the four iron atoms, and the iron in thiscluster is bound to the protein skeleton via fourcysteinyl sulfurs. It is assumed that the Fe4S4*centers of other ferredoxins and the iron-sulfur-containing enzymes have a similar structure be-cause of their similarity in optical and magneticproperties.The Fe4S4* clusters are thought to have three

possible oxidation states in biological systems(the three-state hypothesis) (55, 118, 121). In itsmost oxidized state, (Fe4S4*S4')l, the center isparamagnetic and is representative of the oxi-dized HiPIP center, it exhibits a relatively fea-tureless symmetric EPR spectrum with a reso-nance peak nearg = 2.01 (Fig. 2). A one-electronreduction of the most oxidized form of the clus-ter results in (Fe4S4*S4")I, which is diamag-netic and therefore EPR silent; this center isindicative of the reduced HiPIP and oxidizedclostridial-type ferredoxins. A further one-elec-tron reduction generates (Fe4S4*S4)3-, whichis paramagnetic and representative of reducedclostridial-type ferredoxins; this center has amajor resonance peak near g = 1.94 (see EPRspectrum of Bacillus stearothermophilus ferre-doxin [Fig. 2]). Evidence in support of the three-state hypothesis is as follows: (i) the reduced(Fe4S4*S4C)I diamagnetic cluster of Chroma-tium HiPIP could be further reduced to a para-magnetic (Fe4S4*S4)3- state with the typical g= 1.94 EPR spectrum (48); and (ii) Sweeney etal. (264) observed that oxidation of the predom-inantly diamagnetic (Fe4S4*S4)2- clusters ofclostridial ferredoxin with ferricyanide produceda superoxidized iron-sulfur cluster with an EPRsignal at g = 2.01, indicative of the -1 valencestate. Both experiments (which involved unfold-ing of the Fe-S proteins in solvents containingdimethyl sulfoxide) clearly demonstrated thatthe tetranuclear Fe-S cluster can exist in threeoxidation states.When Carter et al. (55) first proposed that the

Fe4S4* cluster might exist in three oxidationstates, it was thought that the electronic transi-tion between the -1 and -2 oxidation statesoccurred only at relatively high redox potentials,whereas the transition between -2 and -3 statesoccurred only at low potentials. Recent evidencefrom studies on succinate dehydrogenase (197),Azotobacter vinelandii ferredoxin I (265), andthe Desulfovibrio gigas ferredoxins (50) indi-cates that one cannot predict from the opera-tional valence states of the iron-sulfur clusterthe potential range over which these oxidation-


g value2.2 2.0 1.8II I I I~~~~~~~~~~~~

2121111_lii,fll11110.30 0.34 0.38 0.40

Magnetic field MT)FIG. 2. EPR spectra representative of the various

types of bacterial iron-sulfur centers (modified fromHall et al. [113]). Chromatium HiPIP was in theoxidized state, and the other iron-sulfurproteins werein reduced state; all EPR measurements were atabout 20°K.

reduction transitions occur. This suggests thatthe protein moiety determines not only whetherthe iron-sulfur center cycles between the -1 --2 or -2 =-3 oxidation states, but also deter-mines the redox potential at which these tran-sitions occur.As Table 2 shows, the bacterial ferredoxins

that contain Fe4S4* centers are found in a largenumber of microorganisms. In this table, theferredoxins are divided into electron carrierswith (i) a single Fe4S4* cluster, (ii) two Fe4S4*clusters that function at the same oxidation-re-duction potential, and (iii) two or more Fe4S4*


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388 YOCH AND CARITHERS

TABLE 2. Characteristics of ferredoxins which contain Fe4S4* clustersFerre- No. of

Source doxin des- clus- Em (mV) Redox transition` Mol wt Reference(s)ignation ters

Ferredoxins with a single Fe4S4* clus-ter

Desulfovibrio gigas (monomer) 1 NDa -2 -3 6,200 152Bacillus stearothermophilus 1 -280 -2 -3 8,000 188Bacillus polymyxa FdI 1 -390 -2 -3 9,000 3, 202, 234,

261, 321Bacillus polymyxa FdII 1 -422 -2 -3 9,000 261, 310Clostridium thermoaceticum 1 ND -2 -3 7,300 308Desulfovibrio desulfuricans 1 -330 -2 -3 6,000 322Mycobacterium flavum FdII 1 ND -2 -3 ND 6Rhodospirillum rubrum FdII 1 -430 -2 -3 14,500 239, 315Spirochaeta aurantia 1 ND -2 -3 6,000 131Chromatium vinosum HiPIP 1 +350 -1 -2 9,850 15, 54Paracoccus sp. HiPIP 1 +360 -1 -2 ND 124, 269

Ferredoxins with two Fe4S4' clustersthat function at the same EmChlorobium limicola FdI & II 2 ND -2 -3 7,000 267, 268Chromatium vinosum 2 -480 -2 -3 10,000 12Clostridium acidi-urici 2 -434 -2 -3 6,000 45, 262Clostridium pasteurianum 2 -390 -2 -3 6,000 45, 206, 266Peptococcus aerogenes 2 -427 -2 -3 6,000 38, 262

Ferredoxins with two or more Fe4S4*clusters that function at differentEm's

Rhodospirillum rubrum FdI 2 ND -2 -3 14,000 239, 315Azotobacter vinelandii FdI 2 +320, -420 -1 -2 14,500 265, 313, 318Mycobacterium flavum FdI 2 +230, -420b -1 -2 11,200 6Rhodospirillum rubrum FdIV 2 +355, -380 -1 -2 14,000 319Desulfovibrio gigas FdI 3 -455 -2 -3 >> -1 -2 18,000 40, 50, 152Desulfovibrio gigas FdI 3 -430, -30 -2 = -3 =-1 -2 18,000 40, 50Desulfovibrio gigas FdII 4 -130 -2 -3 < -1 -2 24,000 40, 50

" ND, not determined.'M. G. Yates and H. Bothe, personal communication.See Addendum in Proof (p. 410).

clusters that operate at different redox poten-tials.

Ferredoxins with a single Fe4S4* cluster werefirst isolated from D. gigas (152) and Bacilluspolymyxa (202, 241, 321). The occurrence of asingle Fe4S4* cluster (instead of two Fe2S2* clus-ters) in four-iron ferredoxins was demonstratedby Stombaugh et al. (261), who showed that, onfull reduction, B. polymyxa ferredoxin took upone electron per molecule (two electrons wouldbe required to reduce a four-iron protein con-taining two Fe2S2* centers). These single-clusterferredoxins generally have molecular weights be-tween 6,000 and 10,000. From a comparison ofthe physical properties of these iron-sulfur pro-teins with those of model compounds, it wasproposed that these ferredoxins cycle betweenthe (Fe4S4*S4CYs)2- and (Fe4S4*S4CYS)3- oxidationstates at potentials near -400 mV (118, 121).The Chromatium HiPIP is an example of a

class of four-iron electron carriers that cyclesbetween the -1 - -2 oxidation states at arelatively high potential (+350 mV). Evidencefor these redox transitions comes from the ob-servation that oxidized HiPIP is paramagnetic(suggesting a -1 valence), whereas reduced

HiPIP is diamagnetic (suggesting a -2 valence)in the reduced state (15). Taken with the modelstudies on Fe4S4(SR)4 synthetic analogs (118,121), the evidence for the -1 =-2 redox tran-sitions in Chromatium HiPIP is quite compel-ling. X-ray crystallography data showing thatthe Fe4S4* cluster of Chromatium HiPIP (54) isstrikingly similar to the Fe4S4* clusters of P.aerogenes ferredoxin (246) again suggest thatthe peptide determines both the oxidation stateand the operational redox potential of the Fe4S4 *cluster. In contrast to the vast amount of evi-dence that has been compiled on the physicaland chemical characterization of ChromatiumHiPIP, there is little or no evidence as to itsbiochemical role in the cell.A number of ferredoxins normally referred to

as clostridial-type ferredoxins have two Fe4S4*clusters which function at about the same neg-ative redox potential. The two Fe4S4* clusters ofClostridium acidi-urici ferredoxin were shown,for example, to differ in potential by less than 10mV (209). These ferredoxins are found primnarilyin anaerobic bacteria such as the clostridia andthe photosynthetic bacteria; here they catalyzenumerous two-electron transfer reactions, such

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as hydrogen evolution and pyridine nucleotidereduction.

All of the known ferredoxins which functionbetween the -2 - -3 oxidation states havemolecular weights of 6,000 to 9,000. The compli-cated nature of the 8Fe-8S* C. pasteurianiumferredoxin EPR spectrum compared with that ofthe 4Fe-4S* B. stearothermophilus ferredoxin(Fig. 2) is believed to be a result of spin couplingbetween the two Fe4S4* clusters of the former(172).The arrangement of the two Fe4S4* clusters in

relation to the peptide in a clostridial-type fer-redoxin has been determined by X-ray crystal-lography (3, 246) (Fig. 3). The protein backboneof the ferredoxin from P. aerogenes is folded insuch a way that each of the Fe4S4* clusters,which are about 1.2 nm apart, is bound by cys-teine residues from both ends of the polypeptide;that is, cysteines 8, 11, 14, and 45 are coordinatedto iron atoms in one complex, and cysteines 18,35, 38, and 41 are coordinated to the iron in thesecond complex (3). The position ofthe cysteinylresidues in ferredoxins from both fermentingand photosynthetic bacteria is rigorously con-served (309), suggesting a similar environmentfor the Fe4S4* clusters of these ferredoxins.The ferredoxins with two iron-sulfur clusters

that operate with different redox potentials be-long to the third class of bacterial ferredoxins.These two-cluster ferredoxins are found inaerobes such as A. vinelandii (265) and Myco-bacterium flavum (M. G. Yates and H. Bothe,personal communication) and are referred to

2 3 4 5 6 7Ala -Tyr- Vol -Ili1-Asn-Asp-Sor-

8 9 t0 II lz 13 14 35Cys-ll u-Ala-C ys-B1 y-Ala-Cys-

33315 16 17 Is 19 20 21L Ys-Pro-Glu-Cya-Pro-Val-Asu-

22 23 24 25 26 27 31lIu- GIu-Glu-61y-Ser-liu-

20

28 29 3C 31 32 33 34Tyr-Alo-Ilu-Asp-Ala-Asp-S r-

2235 36 37 38 39 40 41C y -I- u- Asp-Cys-Gl y-Ser-Cyss-

21

42 4! 44 45 46 47 48Ala- Sr-Va l-Cys-Pro-Val-Gly-

49 50 Cl 52 53 54Ala-Pro-Asn-Pro-Glu-Asp

here as Azotobacter-type ferredoxins. Unlikeother 8Fe-8S* ferredoxins, these proteins exhibita single EPR absorption band in the oxidizedstate at g = 2.01 (Fig. 4), which is now taken tobe indicative of Fe4S4* centers which functionbetween the -1 =-2 oxidation states, analo-gous to HiPIP. Electron spin quantitation bySweeney et al. (265) indicated that only one ofthe two Fe4S4* centers was functional at lowpotential (Em = -420 mV). When one electronwas taken up, the g = 2.01 EPR signal of thislow-potential center disappeared. The secondFe-S center of Azotobacter ferredoxin I wasobserved by optical and EPR spectroscopy onlyafter oxidation of the protein with ferricyanide(265). This center, which is in the reduced statein the isolated protein, showed a positive Em of+340 mV after equilibrium of the protein withthe ferro-ferricyanide couple. This high-poten-tial center, which was EPR silent (i.e., reduced)in the isolated protein, also had a g = 2.01 EPRsignal, indicating that it also functions betweenthe -1 =-2 oxidation states. The widely diver-gent Em's of the two Fe-S clusters of A. vinelan-dii ferredoxin have been shown by a potentio-metric titration to differ by almost 750 mV (Fig.5). In this titration (monitored optically), thehigh-potential center was preoxidized with fer-ricyanide and then titrated reductively (318).The observations cited above (265, 313) now

explain earlier data which showed that A. vine-landii ferredoxin I transferred only one electron(n = 1) at -420 mV (313) and which drewcriticism (199) based on the then prevalent er-

FIG. 3. Structure of Peptococcus aerogenes ferredoxin showing the arrangement of the two Fe4S4* clusterson the peptide (from Adman et al. [3]).

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NativeA vinela/did

fweradoKnn 12

g-2. 01

300 3200 3400Fied (Gauss)

FIG. 4. EPR spectrum of Azotobacter vinelandiiferredoxin I as it is isolated. The EPR signal is fromthe low-potential Fe4S4* center which is paramag-netic in the oxidized state; the high-potential Fe4S4*center is reduced (diamagnetic) here and therefore isnot contributing to the EPR spectrum.

roneous beliefs that all ferredoxins with twoiron-sulfur clusters should transfer two low-po-tential electrons and that titration data fromthese ferredoxins should fit a Nernst equation inwhich n = 2.The unusual characteristics of the Azotobac-

ter-type ferredoxins, compared with clostridial-type ferredoxins, may be due to a difference inthe basic protein structure. Such a differencewas first suggested by experiments showing thatAzotobacter ferredoxin I and C. pasteurianumferredoxin were denatured at different rates byguanidine and sulfhydryl reagents (313). Al-though all cysteine residues in the amino-ter-minal sequence of the Azotobacter ferredoxincorrelate with homologous residues in other fer-redoxins, the appearance of a cysteine residue inposition 24 is believed by Howard et al. (125) tocause a major change in the environment of oneof the two iron-sulfur clusters. The unusual re-dox characteristics of A. vinelandii ferredoxin Imay result from this variation in ligand arrange-ment.The role of Azotobacter-type ferredoxins in

cellular metabolism is not well understood atthis time. Although the low-potential Fe4S4*center ofthis ferredoxin type functions in typicalferredoxin-requiring reactions, such as nitrogenfixation, the role of the high-potential centerremains unknown. A functional role for this typeof ferredoxin became even more obscure whena similar ferredoxin was isolated from chromat-ophore membranes of the photosynthetic bac-

terium Rhodospirillum rubrum (319). Thus,whatever the role(s) of this unusual ferredoxinin bacterial metabolism, it is not, as first be-lieved, restricted to reactions peculiar to theaerobic bacteria.

Finally, the Fe4S4* ferredoxins of D. gigaspresent another unusual situation. In these fer-redoxins (three have been isolated), the basicunit is a single polypeptide chain of approxi-mately 6,000 daltons, which supports one Fe4S4*cluster (40). This basic unit associates to formthree different ferredoxins; ferredoxins I and I'are trimers of the 6,000-dalton subunit and fer-redoxin II is a tetramer. The interaction of thepolypeptides in the trimer and tetramer pro-duces proteins in which the Fe4S4* clusters ex-hibit different oxidation transitions and differentEm's (50) (Table 2).The trimer, ferredoxin I from D. gigas, gives

a strong g = 1.94 EPR signal on reduction (Em= -455 mV) and a much weaker signal at g =2.01 in the oxidized state, a pattern similar tothat of the Bacillus ferredoxins. The other tri-mer, ferredoxin I', however, shows EPR signalsof about equal intensity at both g = 2.01 (Em =-30 mV) and g = 1.94 (Em = -430 mV), whichindicates that about an equal number of iron-sulfur centers are functioning between both -1=-2 and -2 =-3 transitions. Finally, thetetramer, ferredoxin II, was shown on reductionto exhibit an EPR signal of high intensity at g= 2.01 (Em = -130 mV), with a much weakersignal atg = 1.94. The unusual characteristics of

-500 -400 -300potential (m)

.300 +400

FIG. 5. Potentiometric titration of the high- andlow-potential Fe4S4* clusters ofAzotobacter vinelan-dii ferredoxin I. Symbols: 0, change in absorbanceat 510 nm (AA510 )-reductive titration with ascor-bate ofthe high-potential Fe-S center which had beenoxidized by pretreatment with 60 gLM ferricyanide;0, change in absorbance at 410 nm (AA410 .)-reduc-tive titration with dithionite of the low-potential Fe-S center. Both titrations were carried out anaerobi-cally, and the reaction mixtures contained redoxmediator dyes appropriate for the potential rangeover which the titration was conducted. A Pt-AglAgCl electrode in the titration cuvette permitted thesimultaneous measurement of the absorbance andthe oxidation-reduction potential (from Yoch andCarithers [3181).

0.01OA * OOO4AA

I JL~~~

Em-424mV J m32OlVna, no,

. 0 .. . Im^^ A^^

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the D. gigas ferredoxins have been explained by"the possibility that changes in quatemarystructure of the different oligomers can affectthe relative stabilities of the (iron-sulfur) oxida-tion states of the same monomeric unit" (50). Incomparing the E. of Chromatium HiPIP (+350mV) with that of D. gigas ferredoxin II (-30mV) (both of which function between the -1=-2 redox states), it is again obvious that thepolypeptide both stabilizes the oxidation statesand has a direct influence on the redox potentialof the Fe4S4* cluster.

IRON-SULFUJR ENZYMESThe iron-sulfur cluster (in an appropriate lig-

and environment) is one of the most reducingredox centers found in nature, and for this reasonit is very often found as the prosthetic group inoxidoreductases where the oxidized/reducedcouple of the substrate is below 0 mV. Thus, themetabolism of numerous substrates, from acet-aldehyde and a-keto acids, which have reductionpotentials around -550 mV, through succinatewith a reduction potential near 0 mV, is cata-lyzed by enzymes containing iron-sulfur centers.However, just as there are a small number ofhigh-potential iron-sulfur electron carriers, sotoo are there iron-sulfur-containing enzymes (in-volved in bacterial nitrate and nitrite metabo-lism) that function in this positive redox range.Because a large number of bacterial iron-sul-

fur enzymes have now been isolated and areunder active study, review of this subject mustin some way be restricted. The discussion ofthese enzymes is therefore limited to a brief


description of their cellular functions and bio-chemical characteristics and the new develop-ments in the field. The treatment given to eachenzyme generally reflects the amount of infor-mation available on that particular enzyme; insome cases, so little is known about the enzymethat one can little more than catalog its existenceas an iron-sulfur-containing enzyme. The iron-sulfur enzymes have been arbitrarily groupedinto several classes depending upon the natureof their electron-carrying prosthetic groups. AsTable 3 shows, the least complex iron-sulfurenzymes contain no additional cofactors,whereas the most complex enzymes of this groupcontain the iron-sulfur complex plus three ad-ditional cofactors.

Iron-Sulfur EnzymesHydrogenase. Hydrogenase, perhaps the

most extensively studied of all the simple iron-sulfur enzymes, is found among all the majorphysiological groups of bacteria, including theaerobic hydrogen bacteria, anaerobes, photosyn-thetic bacteria, cyanobacteria, and rhizobiumroot nodule bacteroids (180). The function ofhydrogenase in anaerobes is to provide a type ofanaerobic respiration in which excess reducingequivalents are transferred to protons with thesubsequent evolution ofhydrogen. These hydro-genases are bidirectional in that they catalyzeboth the production and the oxidation of molec-ular hydrogen. In contrast, H2 uptake hydrogen-ases (which are unidirectional, H2-oxidizing en-zymes only) are found in the aerobic hydrogenbacteria (234), where they provide the cell with

TABLE 3. Bacterial iron-sulfur enzymesProsthetic group

Iron-sulfurEnzymes

Hydrogenase, -hydroxylase, 4-methoxybenzoate-0-de-methylase, iron-sulfur proteins bound to procaryoticphotosynthetic membranes, glutamine phosphoribosylpyrophosphate amido transferase

Iron-sulfur-thiamine pyrophosphate ............ Pyruvate-ferredoxin oxidoreductase

Iron-sulfur-flavin. Succinate dehydrogenase, NADH dehydrogenase, Pseu-domonas formate dehydrogenase, dihydroorotate de-hydrogenase, glutamate synthase, adenylyl sulfate re-ductase, trimethylamine dehydrogenase

Iron-sulfur-heme Sulfite reductase (dissimilatory)

Iron-sulfur-molybdenum. Nitrogenase, nitrate reductase (dissimilatory), CO2 reduc-tase (formate dehydrogenase), iron-sulfur-molybdenumprotein of unknown function

Iron-sulfur enzymes with two or more additionalcofactors .................................. Sulfite reductase (asimilatory), coliform formate dehy-

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reducing equivalents and initiate energy-yield-ing processes which permit autotrophic growthon C02. In the nitrogen-fixing cyanobacteria(270), photosynthetic bacteria (32), Azotobacter(252), clostridia (57), and Rhizobium bacteroids(156) the uptake hydrogenase is believed to re-cycle the H2 by-product of nitrogenase as ameans of conserving energy (74, 75).Depending upon the electron carrier with

which hydrogenase reacts, the enzymes may beclassified as follows:

(i) Hydrogen dehydrogenase (EC 1.12.1.2)

NADH + H+ NAD+ + ½H2

(ii) Cytochrome c3 hydrogenase (EC 1.12.2.1)cytochrome C3 (Fe II) + H+

=cytochrome c3 (Fe III) + 1/2H2

(iii) Ferredoxin hydrogenase (EC 1.12.7.1)

ferredoxinr + H+ = ferredoxin0x + 1/2H2In addition, Adams et al. (2) have shown thatclostridial hydrogenase can be reduced by a syn-thetic tetranuclear analog of ferredoxin.Although the bidirectional hydrogenase from

C. pasteurianum has been studied in consider-able detail, there are a number of contradictoryreports concerning this enzyme, not all of whichhave been resolved. Most workers agree that theclostridial hydrogenase is linked to ferredoxin(58, 111) and that the molecular weight of thenative enzyme is 60,000 (58, 88, 191, 192); thenumber of subunits, however, remained in doubtuntil Chen and Mortenson (58) showed that theenzyme was a single polypeptide. The numberof atoms of iron and sulfide per mole of enzymehas remained an open question. Nakos and Mor-tenson (191) and Erbes et al. (88) reported thatthe enzyme contained four nonheme irons andfour acid-labile sulfurs; consistent with thesevalues were the observations that the thiophenolextrusion product of hydrogenase was derivedfrom Fe4S4* clusters (88, 105). Although there isgeneral agreement that C. pasteurianum hydro-genase contains Fe4S4* clusters, there is widedisagreement on the number of these centersper molecule. This disagreement arises from thecontrasting reports of Erbes et al. (88), whosehydrogenase preparation contained 4 Fe and 4S per molecule, and Chen and Mortenson, whoreported 12 Fe and 12 S per molecule. Quanti-tative extrusion of the Fe4S4* cores from this12Fe-12S* preparation indicated that each mol-ecule had approximately three of these tetra-meric centers, consistent with the higher Fe-Scontent of their preparation (105). It is believedby these workers that the iron and sulfur of

clostridial hydrogenase exists as three Fe4S4*clusters (59, 105).

Clostridial hydrogenase exhibits EPR signalsin both the reduced (g = 2.079, 2.007, 1.961,1.936, 1.908, and 1.892) and oxidized (g = 2.099,2.046, and 2.005) forms (59, 88, 192), which areunlike the signals from other known Fe4S4* cen-ters. This complexity may be due to spin-cou-pled centers in the hydrogenase. Because theirEPR data indicated only 1.6 to 1.8 electron spinsper mol of protein, Chen et al. (59) postulatedthat the reduced hydrogenase contains two par-amagnetic (Fe4S4*)3- centers and one diamag-netic (Fe4S4*)2- center.The hydrogenase of another obligate anaer-

obe, Desulfovibrio vulgaris, has also been stud-ied in detail. The Miyazaki strain of this orga-nism has both a soluble and a membrane-boundcytochrome c3-linked hydrogenase (306, 307).The soluble enzyme has a molecular weight of60,000 (306), and the particulate enzyme has amolecular weight of 89,000 (191). Although thenumber of irons in the soluble enzyme is un-known, the particulate species contains seven tonine iron atoms per molecule (307). Like theclostridial hydrogenase, the Hildenboroughstrain NCIB 8303 of D. vulgaris was recentlyreported to contain a 50,000-dalton hydrogenaseconsisting of a single polypeptide chain with 12atoms each of nonheme iron and acid-labile sul-fur per molecule (295). Because this hydrogenasecould be removed from the cell without disrupt-ing the cell membrane, it is believed to be locatedoutside the cytoplasmic membrane, i.e., peri-plasmic (19).A similar periplasmic, cytochrome c3-linked

hydrogenase has also been isolated from D. gi-gas (116). Its molecular weight is 89,500, and itis composed of two different subunits of molec-ular weights 62,000 and 26,000. It contains 12atoms each of iron and sulfide, and, similar tothe clostridial hydrogenase, quantitative extru-sion of its Fe-S centers indicated three Fe4S4*clusters (116).The hydrogenases of the photosynthetic bac-

teria R. rubrum (1, 107) and Thiocapsa roseo-persicina (108) have both been reported to bebidirectional enzymes containing four Fe andfour S* and to have molecular weights of about65,000. Although the activity of the R. rubrumenzyme is rather low, it has been shown tocouple to ferredoxin in both H2 uptake and H2evolution reactions (1, 107). The bidirectionalnature of this enzyme is surprising in light of thefact that the R. rubrum enzyme was alwaysassumed to function only as an uptake hydro-genase (32).The chromatophore-bound hydrogenase from

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C. vinosum can apparently reduce ferredoxin(98), but loses this ability after it is solubilizedby detergent (106,135). Gitlitz and Krasna (106),who first solubilized the enzyme, reported a mo-lecular weight of about 100,000, with two 50,000-dalton subunits. Kakuno et al. (135), however,found a molecular weight of only 70,000 and avariable subunit composition which dependedon the sodium dodecyl sulfate concentration.The Chromatium hydrogenase contained fourFe and four S* (106, 135), and one group (135)suggested that a flavin peptide may be associ-ated with it. The loss of ferredoxin-reducingactivity and the unusual EPR characteristics(106) of Chromatium hydrogenase suggest thepossibility that the purified form may no longerbe in its native state.An unusual characteristic of the hydrogenases

from photosynthetic bacteria is that they appearto be relatively insensitive to 02, compared withthe bidirectional hydrogenases of other obligateanaerobes. Furthermore, although these hydro-genases are bidirectional in vitro, they appear tofunction primarily as uptake hydrogenases invivo (311).The unidirectional uptake hydrogenases, as a

group, are not as well understood as the bidirec-tional hydrogenases. In the hydrogen bacteriathe unidirectional hydrogenases play a criticalrole in the autotrophic life of the organisms.Among this group, various Hydrogenomonasspecies have long been known to couple H2oxidation directly to the reduction of nicotin-amide adenine dinucleotide (NAD) (204, 303)for use in C02 fixation. The electron-carryingprosthetic group of the hydrogenases from hy-drogen bacteria has remained unknown untilrecently, but recent evidence from at least onespecies, Alcaligenes eutrophus, indicates that itis not an iron-sulfur center, but riboflavine 5'-phosphate (FMN); 2 mol ofFMN was recoveredper mol of hydrogenase (235). A newly discov-ered uptake hydrogenase from the anaerobe C.pasteurianum is located in the periplasmic spaceand, like the bidirectional enzyme, it is reportedto couple to ferredoxin; its prosthetic group re-mains unknown (57).Models of catalytic action by hydrogenase

which have been proposed are based upon thecharacteristics of the clostridial hydrogenase. Inone view (88), the enzyme is thought to have asingle Fe4S4* cluster which cycles between afully oxidized state (with a rhombic EPR signal),a partially reduced state (EPR silent), and afully reduced state (with a complex EPR signal)containing a single electron spin. In a differentmodel (59), there are several Fe4S4* centers andanother unknown center (called X) which is

believed to have a more negative Em than thefully reduced iron-sulfur centers, and it is thiscenter X which is thought to be responsible forreducing the protons to hydrogen.w-Hydroxylase. The formal name of this

iron-sulfur enzyme is alkane (or fatty acid), re-duced NAD(P):oxygen oxidoreductase (hydrox-ylating), and it occurs in a number of aerobicorganisms capable of metabolizing alkanes (176).The enzymatic hydroxylation of alkanes byPseudomonas oleovorans has been studied ex-tensively and was shown to require both a re-ductant and 02 as substrates. Rubredoxin func-tions as an electron carrier coupling electronsfrom reduced NAD (NADH) via a flavoproteinNADH-rubredoxin reductase to the hydroxylase(214) (Fig. 6). The enzyme from P. oleovaransis a soluble, 2 x 106-dalton protein composed of42,000-dalton subunits (175). Except for tracesofflavine adenine dinucleotide (FAD), the activeredox components appear to contain approxi-mately four nonheme iron atoms per 2 x 106daltons; these iron atoms exhibit EPR signals atg = 4.3 (rhombic iron signal) and g = 1.94 (theiron-sulfur signal) and a relatively strong signalat g = 2.003 (175).4-Methoxybenzoate-0-demethylase. The

biodegradation of aromatic O-alkyl (phenolic)esters as a source of carbon and energy is gen-erally initiated by monooxygenase systems re-quiring NAD(P)H and 02. In P.putida inductionof this demethylase by 4-methoxybenzoic acidallows the organism to grow on this and otherO-alkyl compounds (26). The enzyme is not veryspecific with regard to the substrate, in that itdealkylates a number of diverse molecules. Fur-thermore, in the presence of NADH and 02 itattacks and opens the aromatic ring (24).

In P. putida a 4-methoxybenzoate-O-demeth-ylase has been isolated as a soluble enzymecomplex which consists oftwo different proteins;the first is an iron-sulfur flavoprotein, and thesecond (the terminal oxidase) is a simple iron-sulfur enzyme. The 42,000-dalton iron-sulfur fla-voprotein contains FMN and an iron-sulfur cen-ter, is reduced by NADH, and functions as thereductase for the iron-sulfur enzyme (26, 28, 40).The iron-sulfur-containing demethylase acceptselectrons from the reductase and functions as

CH -RNADH Reductase Rlihrcdoxi (/ (ox) (Fe") \ O

)( )t (v-h\ dro\\ Id1ic

Reductase Ruhbredoxjn 0NAIl) (red) (Fc) (QHH(OCHH -R

FIG. 6. w-Hydroxylase system of Pseudomonasoleovorans (after Peterson et al. [214]).

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the terminal oxidase. It is a 120,000-dalton pro-tein dimer containing an Fe2S2* center which onreduction with NADH and the reductase ex-hibits EPR signals at g = 2.01, 1.91, and 1.78(28). In its EPR characteristics it closely resem-bles the Rieske protein (225) except that the Emof this center in the oxidase is reported to beabout +5 mV (27), which makes it about 270 mVmore reducing than all other proteins containingReiske-type Fe-S centers. This iron-sulfur-con-taining demethylase not only represents a newsubgroup of the oxygenases, but it is the firstprotein in which substrate-induced reduction ofa Rieske-type iron-sulfur center has been ob-served.

Iron-sulfur proteins bound to procar-yotic photosynthetic membranes. Iron-sul-fur proteins, because of their lack of uniqueoptical features in membranes, have only re-cently been discovered as important constitu-ents of the highly pigmented (chlorophyll-con-taining) photosynthetic membrane. It has onlybeen since the application of low-temperatureEPR spectroscopy to photosynthetic systemsthat it has been possible to detect iron-sulfurcenters in chlorophyll-containing membranes.EPR analysis of membrane-bound iron-sulfur

proteins was first applied to chromatophoresfrom the purple sulfur bacterium Chromatium.These membranes showed two iron-sulfur cen-ters, which, in the reduced state, had g valuesnear 1.94 (78, 94) and could be distinguishedfrom one another on the basis of their oxidation-reduction potentials (one iron-sulfur center hadan Em of -50 mV and the other had an Em of-290 mV) (94). These centers have not yet beendemonstrated to undergo light-dependent oxi-dation or reduction reactions, nor is there anyinformation available as to a possible enzymaticactivity involving these iron-sulfur centers.

In contrast to the two g = 1.94 centers inChromatium, the green sulfur bacterium Chlo-robium and all of the purple nonsulfur bacteriahave three such iron-sulfur centers which arealso distinguished from one another on the basisof their midpoint oxidation-reduction potentials.An example of a typical titration used to resolve

%2

NN..

Za

to

I I I I I I

WooEm: -390 mV

~~I160 -

- Emz -175 mV

120-

80 0

40

Em4+20mV0 l

W ftf - -0 -zoo -400Eh (mV)

FIG. 7. Oxidation-reduction titration of the g =1.93 signal in Rhodospirillum rubrum chromato-phores. Oxidation-reduction titrations of the mem-brane-bound iron-sulfur centers were performed byreducing the preparations (containing redox media-tor dyes) with sodium ascorbate or sodium dithioniteand monitoring the extent of reduction (as measuredby the increased amplitude of the g - 1.93 signal) byEPR spectroscopy. Samples were withdrawn from theanaerobic titration vessel at fixed oxidation-reduc-tion potentials (Eh) (as measured by a Pt-Ag/AgClelectrode) and transferred anaerobically to EPRtubes for X-band EPR analysis at 35°K (from Yochet al. [3191).

these iron-sulfur centers is shown in Fig. 7, wherethe amplitude of the EPR signal in R. rubrumchromatophores was monitored at defined oxi-dation-reduction potentials. Table 4 summarizesthe midpoint oxidation-reduction potential ofthe g = 1.94 centers in representative speciesfrom the three families of photosynthetic bac-teria. In R. rubrum these centers may be relatedto succinate dehydrogenase, since titration ofthe solubilized enzyme showed redox centerswith nearly identical Em's (53). There are, how-ever, other enzymes, such as NADH dehydro-

TABLE 4. Oxidation-reduction properties of the g = 1.94-type iron-sulfur centers in chromatophoremembranes ofphotosynthetic bacteria

Family Species E,,, (mV) ReferenceRhodospirillaceae Rhodospirillum rubruma +20, -175, -390 319

Rhodopseudomonas capsulata +30, -235, -335 217Rhodopseudomonas sphaeroides +40, -175, -390 218

Chromatiaceae Chromatium vinosum +50, -290 94Chlorobiaceae Chlorobium limicola -25, -175, -550 146

' In R. rubrum this EPR signal is actually at g = 1.93.

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genase, which are also bound to the chromato-phore and which have EPR and redox charac-teristics very similar to those of succinate de-hydrogenase. It may be necessary to use kineticsor inhibitors to distinguish between the EPRsignals induced by NADH and those induced bysuccinate, since both substrates reduce g = 1.93iron-sulfur center(s) in R. rubrum chromato-phores (119). That NADH dehydrogenase doesreside on the chromatophore is further sug-gested by a distinctive EPR absorption band atg = 2.1 (Yoch, unpublished data), which byanalogy with mitochondria (200) would be as-sociated with centers 3 and 4 of NADH dehy-drogenase.

In 1964, Rieske and co-workers (224, 225) sol-ubilized and purified from bovine heart mito-chondria via Complex III an electron-carryingprotein (molecular weight, 26,000) which con-tained two irons and two sulfides per molecule.This protein is unusual because in its reducedstate it has its major EPR absorption band at gy= 1.89 (g = 2.025, 1.89, and 1.78), which is eitheran extreme value for gave.e or is a representativemember of a new class of iron-sulfur proteins(the other two being the ferredoxin-type g =1.94 and the HiPIP-type g = 2.01 centers). Al-though the function of the Rieske iron-sulfurcenter is unknown, it appears to operate nearcytochrome c in the mitochondrial electrontransport chain.

Several years ago Rieske-type iron-sulfur cen-ters were also observed in chromatophore mem-branes ofboth the purple sulfur bacterium Chro-matium (78, 94) and the purple nonsulfur bac-teria Rhodopseudomonas sphaeroides (218) andRhodopseudomonas capsulata (205), wherethey were characterized by their EPR signal atg = 1.89. The EPR spectrum of the reducedRieske center in chromatophores of R. rubrum(Fig. 8) is representative of those centers foundin other bacterial photosynthetic membranes.The Em of the Rieske center in the purple pho-tosynthetic bacteria ranges from +275 to +310mV. In the green sulfur bacterium Chlorobium,the Rieske center was found to have an Em of+160 mV, approximately 120 mV more negativethan in the purple bacteria.At alkaline pH (beyond pH 6.8 for the Chlo-

robium protein and pH 8 for the R. sphaeroidesprotein) the Em of the Rieske center was foundto be pH dependent. The Em values were shifted-60 mV per pH unit, indicating that at theproper pH this protein can take up one protonalong with the electron on reduction (146). Re-examinations of other Rieske centers, includingthat from mitochondria, have shown a similareffect of pH on the Em (216). These findings


Mentfie field (eeseIFIG. 8. EPR spectrum ofthe oxidized and reduced

Rieske iron-sulfur center in chromatophores of Rho-dospirillum rubrum. Oxidant, Fe(CN6)3-, ferricya-nide; reductant, Q1H2 hydroquinone. EPR measure-ments were at 20"K (Yoch, unpublished data). Eh,Oxidation-reduction potential.

indicate that this carrier may play a role inproton translocation across the membrane, arole suggested for the Rieske protein in mito-chondria even before the pH dependence wasshown (207).Given that the 2Fe-2S* Rieske center may be

a proton carrier, its function in the photosyn-thetic apparatus is still not understood at thistime. Although illumination of Chromatium (94)and R. sphaeroides (218) chromatophores atroom temperature caused the prereduced Rieskecenter to be oxidized, these experiments pro-vided no real indication of its physiological rolein these energy-conserving systems.

Iron-sulfur proteins are also found in the cy-anobacteria, where they are tightly bound to thephotosynthetic membranes and are involved ina plant-type photosynthesis (95). Iron-sulfurEPR signals were first detected in lamellar par-ticles ofAnabaena cylindrica after illuminationat 77°K, a process which oxidized the photosyn-thetic reaction center chlorophyll and reducedthe iron-sulfur center in a manner similar to thatpreviously observed in isolated plant chloro-plasts (170). This treatment resulted in the ap-pearance of a paramagnetic component with an


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EPR spectrum similar to (but not identical to)that of the soluble Fe2S2* ferredoxin of thisorganism and showed g values at g. = 2.05, gy= 1.95, and gz = 1.87 (95). On illumination atroom temperature in the presence of dithionite,additional EPR signals at g = 2.05, 1.93, and 1.90were also generated. In spinach chloroplaststhese two sets of signals were designated centersA and B, respectively, and were shown to beassociated with Photosystem I (170), the pho-tosystem which generates the low-potential re-ducing equivalents. Both iron-sulfur centerswere present in a highly specialized form of bluegreen algae cells called heterocysts (51), whichare known to contain only Photosystem I.The fact that the light-reduced iron-sulfur

center (center A) in chloroplasts is equivalent tothe amount of chlorophyll P700 photooxidized(based on electron spin counting) suggests thatcenter A is the first or primary electron acceptorof Photosystem I. However, at 20°K the photo-reduced iron-sulfur centers A and B in bothchloroplasts and Anabaena fragments (51) re-main in the reduced state when the light isturned off, whereas P700 photooxidation is re-versible, suggesting that P700 is not linked di-rectly to the reduction of centers A and B. Thereis a photo-induced signal, however, withg valuesat 2.08, 1.88, and 1.76 in both chloroplasts andpreparations from the cyanobacterium Chlorog-loea fritschii (89), which is reversible in thedark. This center has been called X and hasbeen proposed to be the primary electron accep-tor of Photosystem I (31, 89). This signal isbelieved to be due to either an iron-sulfur center(91) or an iron-quinone (31) center. At this point,the primary electron acceptor in plant-type pho-tosynthesis (which is found in the cyanobac-teria) remains a matter of controversy.Glutamine phosphoribosyl pyrophos-

phate amido transferase. Glutamine phos-phoribosyl pyrophosphate amido transferasecatalyzes the first amination step in the synthe-sis of purines:Magnesium 5-phosphoribosyl-1-pyrophosphate

+ glutamine = 5-phosphoribosyl-1-amine+ glutamate + magnesium pyrophosphate

Wong et al. (304) have shown recently thatthis enzyme from Bacillus subtilis is a tetramerof 50,000-dalton subunits and that each subunitcontains approximately three irons and two sul-fides. They report that the enzyme is inactivatedby iron extraction or air oxidation, but is notbleached by dithionite (an observation which isat variance with all other iron-sulfur proteins).It was suggested that the iron was organized as

a reduced single iron center and a fully reducedFe2S2* cluster, both of which were thought toserve only as structural components (304). Ad-ditional work is obviously needed to confirmsuch a nonconventional role for an Fe-S center.

Iron-Sulfur-Thiamine PyrophosphateEnzymes

Pyruvate dehydrogenase. The iron-sulfurenzymes that contain thiamine pyrophosphate(TPP) are involved in the reduction of ferre-doxin by pyruvate and similar a-keto organicacids. The most thoroughly characterized ex-ample of these enzymes is the clostridial pyru-vate-ferredoxin oxidoreductase (EC 1.2.7.1). Inthe saccharolytic clostridia, such as C. pasteur-ianum and Clostridium butyricum, pyruvate de-hydrogenase serves as one of the key enzymes(along with glyceraldehyde phosphate dehydro-genase and NADH/NADH-ferredoxin reductase[134]) in providing glucose-fermenting cells withreduced ferredoxin for N2, proton, C02, and nic-otinamide adenine dinucleotide phosphate(NADP) reduction and acetyl coenzyme A(CoA) for substrate-level adenosine 5'-triphos-phate (ATP) synthesis. The ferredoxin-depend-ent reductive carboxylation reactions catalyzedby these dehydrogenases functioning in reverseare believed to play a central role in the pho-toautotrophic life of the photosynthetic sulfurbacteria (43) and in providing precursors for thede novo synthesis of several amino acids byrumen bacteria (231).The pyruvate-ferredoxin oxidoreductase from

C. acidi-urici occurs as three isozymes, whichhave molecular weights of about 240,000 andcontain six nonheme irons and three acid-labilesulfurs (289) (the sulfide in the enzyme is, nodoubt, equimolar with the iron). The iron-sulfurcenter(s) of the enzyme is not reduced by pyru-vate without the presence of reduced coenzymeA (CoASH), which suggests an enzyme-boundhydroxyethyl-TPP intermediate (288) and thefollowing sequence of reactions:pyruvate + TPP-E..

= (hydroxyethyl-TPP)-Eo. + C02(hydroxyethyl-TPP)-Eo. + CoASH

= acetyl-CoA + TPP-EredTPP-Er.d + Fd.. = TPP-Eo. + Fdred

Summary: pyruvate + CoASH + Fd..= acetyl-CoA + CO2 + Fdrw

Unlike the other cofactors found in iron-sulfurenzymes, TPP in pyruvate dehydrogenase is in-volved in the formation of the oxidizable sub-

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strate, hydroxyethyl-TPP, but TPP itself ap-pears to have no direct role in the electrontransport reactions.

It has long been assumed that the same en-zyme was responsible for both the forward re-action (the phosphoroclastic splitting of pyru-vate) and the reverse reaction (pyruvate syn-thase) which brings about the fixation of CO2 bya ferredoxin-linked reaction (25). A recent re-port, however, indicates that the two reactionsare catalyzed by different enzymes (230). Thepyruvate- and a-ketoglutarate-ferredoxin oxi-doreductases of other bacteria (5, 22, 47, 93, 141,213, 241) have not been well characterized.

Iron-Sulfur-Flavin EnzymesSuccinate dehydrogenase. Compared with

the mitochondrial enzyme, relatively little workhas been done on bacterial succinate dehydro-genase (EC 1.31.99.1), but this enzyme has beensolubilized from membranes of a number of bac-teria, including Corynebacterium diphtheriae(208), Micrococcus lactilyticus (301), E. coli(143), R. sphaeroides (129), Vibrio succinogenes(150), and R. rubrum (53, 68, 117). The mem-brane-bound succinate dehydrogenase from an-aerobic bacteria serves as a fumarate reductaseand acts as the terminal electron acceptor in ananaerobic electron transport system which gen-erates ATP (149) and produces succinate. Al-though the membrane-bound nature of this en-zyme in photosynthetic bacteria suggests that itfunctions as a fumarate reductase, such a roleseems superfluous in an organism capable ofphotophosphorylation. Nevertheless R. rubrumchromatophores do show a potent fumarate re-ductase activity which is tightly coupled to thehydrogenase, provided a suitable electron carrier(a redox dye) is available (32). Assuming thatthe electron-carrying dye does not provide anartificial link between these two enzymes, nei-ther the native electron carrier coupling theseenzymes nor the physiological significance ofthis reaction is known.Some of the earlier work had suggested that

cytochrome b was a component of bacterial suc-cinate dehydrogenase (197, 208); however, laterstudies indicated that it was similar to the suc-cinate dehydrogenase from mitochondria andcontained only flavin and iron-sulfur centers (53,68, 117, 129, 149, 301). The enzyme solubilizedfrom R. rubrum chromatophore membranes isperhaps the best characterized bacterial succi-nate dehydrogenase. It has a molecular weightof 85,000 (53, 68, 149) and is composed of a60,000-dalton subunit and a 25,000-dalton sub-unit (68, 117). The ratio of flavin to iron tosulfide in the native enzyme is 1:8:8 (68, 149).


Both flavin and nonheme iron are localized onthe larger subunit, whereas the smaller subunitcontains only iron and sulfide (53). The oxidizedenzyme exhibits an HiPIP-type EPR signal froman iron-sulfur cluster (53) which corresponds tothe center S-3 in mitochondria (196). (Iron-sul-fur centers of succinate dehydrogenase are des-ignated with an S, those of NADH dehydrogen-ase are designated with an N, and the numbersdesignate a specific center.) Reduction of theenzyme with succinate reveals another iron-sul-fur center with an EPR signal at g = 2.03, 1.93,and 1.91 (analogous to Fe-S center S-1 in mito-chondria) and a flavin semiquinone signal at g= 2.006 (53, 68, 119). The redox potentials of theFe-S centers of bacterial succinate dehydrogen-ase can be seen from a titration of R. rubrumenzyme (Fig. 9). Three redox centers with Em'sof +50, -160, and -380 mV are observed; thefirst two redox centers probably represent iron-

-500 -400 -300 -ZOO -100 0 00Redc p-otslo/ (n*)

FIG. 9. Oxidation-reduction titration of succinatedehydrogenase from Rhodospirillum rubrum. The en-zyme was solubilized from chromatophore mem-branes with the detergent lauryl dimethylamine oxide(line A) or alkaline (pH 9.5) treatment (line B). Thepotentiometric titrations were performed with an an-aerobic cuvette system that allowed the simultaneousmeasurement of absorbance changes (AA) and oxi-dation-reduction potential changes. The enzyme wastitrated reductively with small additions of 30 mMsodium dithionite. Absorbance changes in the enzymewere monitored at 440 nm (each point was correctedby subtracting absorbance changes of the oxidation-reduction mediator dyes as determined by a priortitration of the mediators alone) (from Carithers etal. [53]).


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sulfur centers S-1 and S-2. The presence of a-380-mV redox center in the solubilized R. rub-rum enzyme suggests a similarity to a mitochon-drial succinate dehydrogenase center character-ized by Ohnishi et al. (197), who observed a-400-mV iron-sulfur component in a solubilizedpreparation. In this beefheart enzyme it appearsthat the -250-mV S-2 center was altered on itsremoval from Complex III and converted to the-400-mV form; on reconstitution with the par-ticulate cytochrome b-c complex the potentialof this Fe-S center shifted back to -250 mV.The existence of both -160-mV and -380-mVcenters in the detergent-solubilized R. rubrumenzyme (53) indicates that this preparation mayhave been heterogenous with respect to the S-2iron-sulfur center. It must be noted, however,that potentiometric EPR titrations of the un-treated membranes also revealed three iron-sul-fur centers with these same redox potentials(Fig. 7).The solubilized succinate dehydrogenase from

R. sphaeroides had EPR characteristics similarto those of R. rubrum (i.e., a flavin free radicaland S-1 and S-2 signals), but only one non-succinate-reducible iron-sulfur center (S-2) wasobserved in a redox titration (129).The enzymatic mechanism of bacterial succi-

nate dehydrogenase is presumed to be the sameas that of the mitochondrial enzyme (144). Ithas been proposed that in this enzyme, electronsflow from succinate through center S-1 and theflavin to center S-3 and from there to the elec-tron transport chain (196, 197). The function ofcenter S-2 (Em = -160 mV) remains unknownin both mitochondrial and bacterial enzymesand may be an artifact of enzyme purification(16, 196, 229), since several groups have reportedvariable values for electron quantitation of thiscenter in the mammalian enzyme. On the otherhand, the occurrence of eight iron atoms(enough for two 2Fe centers and one 4Fe center)and quantitative extrusion oftwo Fe2S2* centers(229) argue against center S-2 of succinate de-hydrogenase being an artifact.Reduced nicotinamide adenine dinucleo-

tide dehydrogenaase. Although NADH dehy-drogenase (EC 1.6.99.3) plays an important rolein energy metabolism, surprisingly little isknown about the bacterial enzyme. Only a cou-ple of bacterial NADH dehydrogenases havebeen isolated. The enzyme from E. coli (111),which was first solubilized by freeze drying mem-branes, is a metalloflavoprotein containing non-heme iron, acid-labile sulfur, FMN, and FAD.The mole ratio of these cofactors was not deter-mined. Recent extensive purifications of the E.coli NADH dehydrogenase have shown it to be

MICROBIOL. REV.

a single polypeptide chain of 38,000 daltons (66),but the flavin cofactor (FAD) was lost duringthe isolation process (66, 67) and again the moleratio of iron-sulfur was not determined.The NADH dehydrogenase from A. vinelan-

dii was first recognized as an iron-sulfur proteinby an EPR signal at g = 1.94, which appearedon reduction of Azotobacter membranes withNADH (17). The enzyme has been isolated intwo forms, depending upon the amount of ironin the growth medium (69). Both forms ofNADH dehydrogenase have molecular weightsof 56,500, but the enzyme isolated from cellsgrown on low levels of iron contained as cofac-tors one molybdenum, two nonheme irons, andtwo sulfides per FMN, whereas the enzyme fromcells grown on high levels of iron contained fourirons and four sulfides per FMN (69). TheNADH-reduced enzyme from low-iron cells ex-hibited a complex EPR spectrum due to Mo(V)(70) and a single iron-sulfur center withg valuesof 2.034 and 1.934 (69). The enzyme from normaliron growth conditions exhibited an EPR signalat g = 2.016 in the oxidized state and signalswith g values at 2.042, 2.034, and 1.940 in thereduced state (69). The mechanism of this en-zyme is unknown.Although the bacterial NADH dehydrogen-

ases appear to have the same general propertiesas the NADH dehydrogenase from mitochon-dna, our detailed knowledge of the Fe-S centersof these bacterial enzymes lags by at least anorder of magnitude. At least part of the reasonmay be the general instability of the bacterialenzymes; also, most interest in this enzyme hasbeen focused, for traditional reasons, on themitochondrial enzyme.Pseudomonas formate dehydrogenase.

Although most formate dehydrogenases aremembrane bound, donate electrons directly toinsoluble electron transport carriers, and havemolybdenum and selenium as additional cofac-tors, the enzyme from Pseudomonas oxalaticusis an iron-sulfur flavoprotein (123) that catalyzesthe following reaction:HCOO- + NAD + H20 = HC03- + NADH + H+

With this enzyme the organism is capable ofgrowing on formate as the sole carbon and en-ergy source (29).The soluble formate dehydrogenase from P.

oxalaticus is isolated in two equally active forms(I and II) having molecular weights of 320,000and 175,000, respectively (122). Sodium dodecylsulfate electrophoresis has shown form I to havetwo subunits with molecular weights of 100,000and 59,000; it contains 2 FMNs and 17 to 20irons per molecule and is assumed to have the


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same amount of sulfide. Formate dehydrogenaseII contains 1 FMN and 8 to 10 irons. The factthat form II was not observed in the early puri-fication steps, but appeared later, suggested thepossibility that form I is a dimer of form II. Theenzymatic mechanism of this iron-sulfur flavo-protein is not known, but the available datashow that for the enzyme to react with eitherformate or NAD(H), it requires the presence offlavin (122).Dihydroorotate dehydrogenase. This en-

zyme catalyzes both the synthesis and degrada-tion of pyrimidines by the following reaction:

L-5,6-dihydroorotate + oxidized NAD

=- orotate + NADH + H+

Clostridium oroticum (formerly Zymobacteriumoroticum) (56), when adapted to ferment oroticacid, synthesizes a high level of the iron-sulfurflavoprotein dihydroorotate dehydrogenase (EC1.3.1.14), and it was from this organism that theenzyme was first purified (9). In the normalcourse of metabolism the enzyme presumablyfunctions mainly in the synthesis of pyrimidines,and this reaction involves an oxidation of dihy-droorotate by oxidized pyridine nucleotidesrather than a reduction of orotate.

Although the enzyme has been isolated fromother organisms, the most thoroughly studiedexample of the enzyme is that from C. oroticum.It has a molecular weight of 115,000 and has theunique property of containing equimolaramounts of FAD and FMN (two each) as wellas four nonheme irons and four acid-labile sul-furs (7, 101, 179). Data from subunit molecularweight determinations and peptide mappingsuggest that the enzyme is a tetramer composedof four identical polypeptide chains (7). It hasbeen proposed that the protein contains twoequivalent catalytic sites and that the iron atomsare grouped in Fe2S2* clusters (7). All of thecofactors seem to participate in the catalysis, asshown by flavin semiquinone EPR signals at g= 2.005 and iron-sulfur signals at g = 2.0, 1.94,and 1.92 in the substrate-reduced enzyme (8, 9).The mechanism proposed for dihydroorotate

dehydrogenase is as follows (7):

NADH (dihydroorotateFAD Fe282* FMN

NAD orotate

such that H(S) on carbon 5 is eliminated (30).Glutamate synthase. Glutamate synthase

(EC 2.6.1.53) catalyzes the following reaction:

glutamine + a-ketoglutarate + NAD(P)H

-= 2 glutamate + NAD(P)+

This enzyme, when combined with glutaminesynthetase, provides a more efficient pathwayfor fixing ammonia than does glutamate dehy-drogenase. Nitrogen-fixing bacteria (193) and anumber of coliforms (169) derepress the synthe-sis of these two enzymes when the level of am-monia in the medium falls below a certain level.Other bacteria, such as Bacillus megaterium(84) and Caulobacter crescentus (85), probablyuse this enzyme system exclusively as a meansof incorporating ammonia into amino acids.The glutamate synthase from E. coli is a

soluble enzyme of 800,000 daltons composed offour 53,000-dalton and four 135,000-dalton sub-units (178). It has been proposed that the en-zyme is actually a tetramer and that the com-bination of one large and one small subunitcontains one FAD, one FMN, eight irons, andeight sulfides (178).The Klebsiella aerogenes (formerly Aerobac-

ter aerogenes) glutamate synthase appears to bea simple dimer composed of a 175,000-daltonsubunit and a 51,500-dalton subunit (280). Thecofactors of this enzyme are reported to be 1FAD, 1 FMN, 7 irons, and 13 sulfides per mole-cule. The large subunit appears to be the bindingsite for glutamine and to contain the cofactors(280). The enzyme is thought to catalyze theamination of a-ketoglutarate without the pres-ence of free ammonia (103) by the followingmechanism (178):H+ + NADPH + E. flav = E. flav. H2 + NADP+

E.flav. H2 + a-ketoglutarate + Gln

= 2 Glu + E.flav + H+Summary: NADPH + a-ketoglutarate + Gln

= 2 Glu + NADP+

Adenylyl sulfate reductase. Some speciesof sulfate-reducing bacteria, such as Desulfovib-rio and Desulfotomaculum, are able to performa type of anaerobic respiration in which theyobtain energy by coupling the oxidation of H2and some organic compounds to the reductionof sulfate. The reducing equivalents are trans-ferred to oxidized sulfur compounds as the ter-minal electron acceptors, with the production ofsulfide. The first step in this process of dissimi-latory sulfate reduction is the reduction of sul-fate (in the form of 5'-phosphoadenylyl sulfate)to sulfite (177). The direct physiological electrondonor to adenylyl sulfate reductase has not beenconclusively established. However, Desulfovib-rio contains ferredoxin, flavodoxin, and cyto-chrome C3, all of which are possible electrondonors; reduced methyl viologen is commonlyused as the in vitro reductant.

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Adenylyl sulfate reductase (EC 1.8.99.2) fromD. vulgaris is an iron-sulfur flavoprotein with amolecular weight of 220,000 and is composed ofone 20,000-dalton subunit and three 72,000-dal-ton subunits (37). The enzyme was determinedto contain 12 irons, 12 sulfides, and 1 FAD, basedon a molecular weight of 220,000. The additionof excess sulfite to an oxidized preparation hav-ing an HiPIP-type EPR signal caused a declinein the g = 2.0 signal, which was accompanied bya parallel rise in a g = 1.94 Fe-S center (212).The number and type of Fe-S clusters (2Fe or4Fe) in adenylyl sulfate reductase are notknown.

In the aerobic sulfur-oxidizing bacteriumThiobacillus denitrificans (35) and in the an-aerobic purple sulfur photosynthetic bacteriumC. vinosum (276), adenylyl sulfate reductase hasbeen shown to function in the reverse direction,that is, in the oxidation of reduced sulfur mole-cules. In both of these organisms the electronsfrom the reduced sulfur molecules are used as asource of reducing equivalents for the auto-trophic fixation of C02. The adenylyl sulfatereductases from Thiobacillus thioparus (211)and T. denitrificans (35) are also iron-sulfurflavoproteins, but the purity of these prepara-tions was apparently not sufficient to allow acomputation of the mole ratio of cofactors toprotein.Trimethylamine dehydrogenase. This en-

zyme has been isolated from a facultative meth-ylotrophic organism where it catalyzes the fol-lowing reaction:

(CH3)3N + H20 + XO. (CH3)2NH + CH20 + Xrd

Trimethylamine dehydrogenase furnishes thismethylotrophic organism with one-carbon unitsfor utilization by either the serine or the ribosephosphate pathway and allows growth on tri-methylamine. The natural electron acceptor forthis enzyme is unknown, but.a flavoprotein hasbeen suggested for this role (257). For in vitroreactions, phenazine methosulfate (a flavin an-alog) is commonly used as the electron acceptor.The enzyme has a molecular weight of 146,800

(257) and contains a single Fe4S4* cluster (120,258) in addition to a yellow organic cofactor.Although the yellow cofactor was originallythought to be a phosphorylated pteridine deriv-ative (258), the most recent evidence identifiesit as a flavin which is substituted in a positionother than in the 8-a-methylene location (259).Chemical reduction of the enzyme with dithi-

onite reveals a simple rhombic EPR signal(g = 2.035, 1.925, and 1.85) due to the iron-sulfurcenter having a single unpaired spin. Substratereduction, in contrast, first generates the quinolform of the flavin, followed by an intramolecular

MICROBIOL. REV.

electron transfer to the iron-sulfur group and anextensive interaction between the flavin semi-quinone spin and the reduced iron-sulfur centerspin (259). The formation of the spin-spin inter-acting species is thought to be the rate-limitingstep in catalysis.

Iron-Sulfur-Heme EnzymesSulfite reductase (dissimilatory). The

"sulfur respiratory" process, which starts withreduction of sulfate to sulfite (see discussion onadenylyl sulfate reductase above), continueswith the reduction of sulfite to trithionite(S3062-) by sulfite reductase, trithionite to thio-sulfate (S2032-) by trithionite reductase, andthiosulfate to sulfide (2-) by thiosulfate reduc-tase (155).The dissimilatory sulfite reductase (EC

1.8.99.1) of Desulfotomaculum nigrificans (for-merly Clostridium nigrificans), which is alsocalled bisulfite reductase because protonatedsulfite is the true substrate, is a brown autooxi-dizable pigment which has been called P582(281). It is a 145,000-dalton protein which con-tains eight atoms of iron, two acid-labile sulfurs,and a siroheme which is an octacarboxylate irontetrahydroporphyrin. Because the molecule con-tains only two sulfide atoms, the release of thesetwo sulfides along with two g atoms of iron permole of enzyme by ferrous iron chelaters sug-gests that the iron-sulfur cluster in this enzymeis of the Fe2S2* type (281). The iron atoms whichare not chelated are responsible for binding theenzyme inhibitor carbon monoxide. Evidencefrom the coliform assimilatory sulfite reductase(243) (see below) suggests that the siroheme ironthat binds CO is also the site of sulfite binding.Akagi (4) has reported that ferredoxin is thephysiological electron donor to this enzyme inD. nigrificans.The respiratory (or dissimilatory) sulfite re-

ductase of Desulfovibrio organisms is called de-sulfoviridin (156); it apparently is not an iron-sulfur protein (155), but it does contain a hemechromophore thought to be sirohydrochlorin(190). D. vulgaris does, however, contain anassimilatory reductase of 23,500 daltons whichhas many properties similar to those of the iron-sulfur-heme respiratory sulfite reductase of D.nigrificans (155), but iron and sulfide have notyet been reported in this enzyme.

Iron-Sulfur-Molybdenum EnzymesThis group of enzymes includes nitrogenase,

clostridial formate dehydrogenase, respiratorynitrate reductase, and a protein of unknownfunction from D. gigas.Nitrogenase. The most important and


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widely studied of the iron-sulfur-molybdenumenzymes is nitrogenase (EC 1.7.99.2). Nitrogen-ase catalyzes the reduction of nitrogen gas toammonia or, in the absence of N2, the ATP-dependent evolution of hydrogen. The distribu-tion of the enzyme is restricted to a relativelyfew species of procaryotes, i.e. Azotobacter, Ba-cillus, Clostridium, Klebsiella, Rhizobium bac-teroids from leguminous plants, Mycobacterium,Spirillum, photosynthetic bacteria, cyanobac-teria, and a few other miscellaneous organisms(324). One is struck by the fact that the nitro-genases from these various sources are remark-ably similar.Although N2 is the normal substrate for nitro-

genase, other compounds, such as nitrites, iso-cyanides, azides, nitrous oxide, and alkynes, arealso reduced (114). One alkyne, acetylene, hasbeen particularly useful in assays for nitrogenasebecause it can be detected by gas chromatogra-phy with great sensitivity (73).

Nitrogenase is composed of two different con-stituents; the smaller component is a simpleiron-sulfur protein called the Fe protein,whereas the larger component is a more complexprotein containing molybdenum as well as iron-sulfur groups and is called the MoFe protein.The number of these constituent proteins innitrogenase is not known with certainty, butratios of Fe protein to MoFe protein of 1:1 (80,81, 238) and 2:1 (23, 183, 284, 294) have beenreported.Fe protein has a molecular weight of 56,000 to

67,000, is composed of two identical polypep-tides, and contains four nonheme irons and fouracid-labile sulfurs (201). The arrangement of theirons into a single Fe4S4* cluster is suggested by(i) the optical spectrum of the thiophenol-ex-truded iron-sulfur cluster (121) and (ii) the de-tection of only one species of iron atom byMossbauer spectroscopy (82, 250). The reducediron-sulfur cluster exhibits an EPR signal at g= 2.06, 1.94, and 1.87 (82, 203, 205) after theuptake of 0.2 to 0.8 electrons (203, 205, 251).Magnesium-ATP alters the characteristics ofthe iron-sulfur cluster of Fe protein in the follow-ing ways: (i) the Em is shifted from -294 to -402mV (325); (ii) the symmetry of the EPR spec-trum becomes more axial (81, 193, 203, 251, 323,326); and (iii) the irons become more sensitiveto an iron chelater (298, 299) and a thiol reagent(278, 285). There are apparently binding sitesfor two magnesium-ATP's on the Fe protein(277, 286, 323, 326).MoFe protein has a molecular weight of

200,000 to 230,000 and is composed of four sub-units (201), which are large enough to be seen inelectron micrographs of the protein (256). In A.vinelandii and most other organisms, the MoFe

protein subunits are present in an a2/32 structure(201), but the protein from Rhizobium japoni-cum is apparently a tetramer of identical sub-units (130). The cofactor content of MoFe pro-tein is not precisely known because preparationsare frequently contaminated with denaturedproteins which have a lowered metal contentand also because analytical errors prevent theabsolute precision needed in quantitating thelarge numbers of iron and sulfides in this protein.Some examples of reported values are shown inTable 5.The organization of these cofactors in MoFe

protein is not precisely known, but there is someinformation available on this point. First, Moss-bauer spectroscopy suggests that the iron atomsare grouped into at least three and possibly fourdistinct units (82,250). Only one center, account-ing for eight iron atoms, changes its propertiesduring turnover of the enzyme (189, 251). Sec-ond, Stasny et al. (256) have suggested fromdata based upon electron micrographs that eachof the four subunits of MoFe protein has anelectron-dense region of heavy metals near itscenter.A more recent approach in studying the dis-

tribution of metals in MoFe protein has been toisolate the enzymatically active cofactor intact(Mossbauer and EPR spectroscopy were used todetermine whether the cofactor was in its nativestate). This iron-molybdenum-containing cofac-tor was isolated as a low-molecular-weight unitcalled FeMoCo (220, 237). Although some of theiron atoms in MoFe protein appear to be inFe4S4* clusters which are distinct from FeMoCo(220, 237), FeMoCo itself contains Fe, S, andMo in a ratio of 8:6:1 such that either MoFe pro-tein has two FeMoCo units and each FeMoCocontains one molybdenum or, alternatively,FeMoCo contains two molybdenums (220).MoFe protein has also been extensively stud-

ied by using EPR techniques. The dithionite-reduced state of the protein exhibits EPR reso-nances near g = 4.3, 3.7, and 2.01 (201), but the

TABLE 5. Cofactor content of nitrogenase MoFeprotein

No. of: Refer-Organism R2efer-Mo Fe S2- ence

Chromatium vinosum 2 14 11 96Clostridium pasteurianum 2 24 24 126

2 24 6 1811 14 16 642 20 20 183

Azotobacter vinelandii 1.5 24 20 1452 24 24 189

Klebsiella pneumoniae 1 17 17 82Rhizobium bacteroids 1.3 29 26 130

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exact value depends upon the origin of the pro-tein and the pH (251). FeMoCo has an EPRspectrum similar to that of MoFe protein (220).Isotope substitutions with 95Mo and 57Fe (96,183, 189, 205, 251) suggest that the EPR reso-nance lines observed in the FeMo protein arisefrom iron atoms and not from the molybdenum.The irons are thought to be arranged in a com-plex which has at least three irons with a netspin of S = 3/2 (189, 205, 251) and contains twounpaired electrons per MoFe protein (189) orone unpaired electron per molybdenum inFeMoCo (220). The reduced state of MoFe pro-tein characterized by the EPR signal at g = 4.3,3.8, and 2.01 is not capable of reducing thesubstrates of nitrogenase, but instead MoFe pro-tein must be reduced further by electrons fromFe protein to produce a "super-reduced" state ofMoFe protein; this reduction is accompanied bya loss of the EPR signals (96, 183, 184, 203, 251,323) and the uptake of four additional electrons(184, 300).The enzymatic mechanism of nitrogenase is

shown in Fig. 10. In this scheme, electrons fromreduced ferredoxin enter nitrogenase on the Feprotein component, which then combines withtwo magnesium-ATP molecules (277, 284, 323,326). The reaction of the reduced Fe proteinwith magnesium ATP causes a shift in the Em ofthe Fe4S4* cluster of Fe protein from -294 to-402 mV (325). The magnesium-ATP-Fe pro-tein complex transfers electrons to MoFe proteinwith the splitting of the -y-phosphate from ATPto given an optimum efficiency of one electrontransferred for two ATPs split (160, 302). Theoperational midpoint oxidation-reduction poten-tial of the MoFe protein is not known withcertainty, but it must be more negative than-260 mV because the EPR signals develop (inpart) with this Em (6), whereas the transition tosuper-reduction exhibits a loss in paramagne-tism. It is tempting to attribute the -460-mV Emobserved for the overall nitrogen-fixing reaction(92) to the super-reduced state of MoFe protein.Nitrate reductase (dissimilatory). Respi-

ratory nitrate reductase (EC 1.9.6.1), like respi-ratory sulfite reductase, is an enzyme used byanaerobic bacteria to dispose of excess reducing

MgADP+Pi

FerredOxinVCd) Fe protein FeMo protein KEN, CH-CH or H+

Ferredoxinox protein FeMo protein 2NH4, C2H4orH2

Mg-ATP

FIG. 10. Mechanism of nitrogenase turnover.ADP, Adenosine 5'-diphosphate; Pi, inorganic phos-phate.

equivalents by depositing them on an inorganicion. Because this enzyme is membrane boundand linked to the electron transport chainthrough cytochromes, the solubilized enzymemay or may not be found to contain hemes as asubunit; we have chosen to include the enzymein this section with the iron-sulfur-molybdenumenzymes because the cytochrome may be anadventitious component.The respiratory-linked nitrate reductase sol-

ubilized from K. aerogenes by deoxycholate ap-pears in two forms, nitrate reductase I and ni-trate reductase II (227). Nitrate reductase I iscomposed of three different subunits of 117,000,57,000, and 52,000 daltons in a ratio of about 1:1:2. Nitrate reductase II is smaller and appears tobe missing the two brown 52,000-dalton subunitswhich have been proposed to couple electronflow to cytochrome bm9 in this organism (227).The cofactors in nitrate reductase I are 0.24 Mo,8 Fe, and 8 S2-, and, in addition, there are fourirons not bound to sulfide (226). These cofactorsare responsible for EPR signals due to Mo(V)and an iron-sulfur cluster(s) of unknown sym-metry (at g = 2.015) in the oxidized enzyme andMo(III) and ferredoxin-like iron-sulfur signals(g = 2.05, 1.95, and 1.88) in the reduced enzyme.

In E. coli the respiratory nitrate reductase hasbeen solubilized by various techniques, and theresulting enzyme has different properties in eachcase (164). The enzyme appears to contain threedifferent subunits; subunit A has a molecularweight of 142,000 and contains the catalytic site,subunit B has a molecular weight of 58,000 andappears to be the membrane attachment site,and subunit C (the cytochrome b peptide), whenpresent, has a molecular weight of 19,500 (86,164, 166-168). Evidence that the B subunit isinvolved in membrane attachment is that solu-bilization of the enzyme by heat treatment ap-pears to be the result of proteolysis, and it is theB subunit which is degraded without loss ofenzymatic activity (164, 166). The necessity forthe cytochrome b component of the E. coli en-zyme is questionable since many of the reportedpreparations do not contain hemes (99, 140, 164,166, 168).

If it is assumed that the core enzyme consistsof one A and B subunit (61, 168), the cofactorcontent from two different preparations hasbeen reported to be 1 Mo, 12 Fe, and 12 S2-(164) and 1.5 Mo, 20 Fe, and 19 S2-, respectively(99). Both the molybdenum and iron-sulfur clus-ters of the enzyme have been studied by EPRtechniques. Oxidized nitrate reductase exhibitsan EPR signal at g = 2.005 due to iron-sulfurclusters and a complex signal nearg = 1.988 dueto Mo(V) (71), but the complexity of the molyb-

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denum signal [presumably due to Mo(V)-,H'hyperfine splitting] was eliminated by recordingthe spectrum in D20, which revealed a simplerhombic symmetry with g values of 1.999, 1.985,and 1.964 (38). Reduction of the enzyme wasaccompanied by a loss of the signal from molyb-denum (38) and the appearance of two differentEPR signals from the iron-sulfur clusters (71).The type I iron-sulfur signal exhibited g valuesof 2.047, 1.889, and 1.861, whereas the type IIsignal had peaks at g = 2.030 and 1.942 (71).The nitrate reductase ofMicrococcus denitri-

ficans has also been solubilized and studied.This 160,000-dalton enzyme contains 0.4 Mo, 8Fe, and 8S2- (100, 153) and exhibits EPR signals(100) similar to those of the E. coli enzyme.CO2 reductase (formate dehydrogenase).

Ferredoxin-dependent formate dehydrogenasein clostridia is believed to function primarily inthe reduction of C02 to formate (273). Becauseof its physiological function, this enzyme is com-monly called C02 reductase or more correctly,ferredoxin-C02 oxidoreductase. Although theenzyme in C. pasteurianum has always beenknown to be extremely sensitive to oxygen (atrait common to many Fe-S proteins), it hasonly recently been isolated and shown to containiron, sulfide, and molybdenum (232). These co-factors occur in a ratio of 24:24:1 based on amolecular weight of 118,000 for the enzyme,which is composed of two subunits with molec-ular weights of 34,000 and 86,000. Although EPRdata are not yet available to implicate the ironand sulfide in the electron transfer process, thereis evidence for the participation of Mo in thecatalytic mechanism, which comes from the ob-servation that both the synthesis and activity ofC02 reductase require the presence of molyb-denum in the growth media (273).

Clostridium thernoaceticum also containsC02 reductase, but unlike the C02 reductase ofC. pasteurianum, the enzyme from this orga-nism uses reduced nicotinamide adenine dinu-cleotide phosphate (NADP) rather than ferre-doxin as a reductant (12). Furthermore, the cellsrequire both molybdenum and selenium for theformation of the enzyme. Although the C02 re-ductase from C. thermoaceticum has been puri-fied (it is composed of several soluble isozymesof about 300,000 daltons), there is, as yet, noindication that it is an iron-sulfur enzyme.Iron-sulfur molybdoprotein of unknown

function. An iron-sulfur molybdoprotein hasbeen isolated recently from D. gigas by Mouraet al. (185); it showed no act

bacterial iron-sulfur proteins · the sulfur atoms in the prosthetic group recog-nizes that the...

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