Heterodisulfide reductase from methanogenic archaea: a new catalytic role for an iron-sulfur cluster

Heterodisulfide reductase (HDR) from methanogenic archaea is an iron-sulfur protein that catalyzes reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol-coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Via the characterization of a paramagnetic reaction intermediate generated upon oxidation of the enzyme in the presence of coenzyme M, the enzyme was shown to contain a [4Fe-4S] cluster in its active site that catalyzes reduction of the disulfide substrate in two one-electron reduction steps. The formal thiyl radical generated by the initial one-electron reduction of the disulfide is stabilized via reduction and coordination of the resultant thiol to the [4Fe-4S] cluster.     

Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme

Heterodisulfide reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe-4S](3+) cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe-4S] cluster using CoM-(33)SH as substrate and purified Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-(33)SH in the presence of duroquinone as an oxidant resulted in a significant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyperfine coupling between the (33)S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe-4S] cluster in the active site of the enzyme.     

Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase

Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A.     

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.     

A paramagnetic species with unique EPR characteristics in the active site of heterodisulfide reductase from methanogenic archaea.

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron-sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and coenzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-electron step with a midpoint-potential of -185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe-enriched enzyme indicates that it is at least partially iron based. The g values (gxyz = 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe-2S]+, [3Fe-4S]+, [4Fe-4S]+ or [4Fe-4S]3+ cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H-S-CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe-4S]3+ cluster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.     

Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophila

Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.     

Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

The properties of the [4Fe-4S] cluster in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been investigated using low temperature magnetic circular dichroism, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies. The Raman spectra of the native enzyme in the Fe-S stretching region show a [4Fe-4S]2+ cluster that is structurally very similar to those in simple redox proteins. Photochemical reduction mediated by 5-deazaflavin with oxalate as the electron donor resulted in [4Fe-4S]+ clusters with a mixture of ground state spin multiplicities. Magnetic circular dichroism and EPR studies of samples ranging in concentration from 0.15 to 0.4 mM concur in finding S = 3/2 [4Fe-4S]+ clusters with predominantly axial and positive zero field splitting as the dominant species. The EPR studies also revealed minor contributions from S = 1/2 [4Fe-4S]+ centers and an S = 5/2 species. The latter becomes the dominant component in more concentrated samples (approximately 2 mM), and arguments are presented in favor of assignment to S = 5/2 [4Fe-4S]+ clusters rather than adventitiously bound high spin Fe(III) ions. The concentration-dependent spin state heterogeneity of the [4Fe-4S]+ cluster in glutamine phosphoribosylpyrophosphate amidotransferase is discussed in light of the magnetic and electronic properties of the [4Fe-4S]+ centers in other enzymes and proteins.     

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions.

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.     

Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe–4S] Cluster Chemistry

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe–2S] ferredoxin as the electron donor and an active site comprising a [4Fe–4S] cluster and a redox-active disulfide. This review summarizes structural and spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) and a chemically modified form, termed NEM–FTR, which provides a stable analog of the one-electron reduced catalytic intermediate. Detailed spectroscopic characterization of FTR and NEM–FTR using absorption, EPR, electron–nuclear double resonance, variable-temperature magnetic circular dichroism, resonance Raman and Mössbauer spectroscopies indicate that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation of a [4Fe–4S]²⁺ cluster to yield a unique type of S= 1/2 [4Fe–4S]³⁺ cluster with two cysteine residues ligated at a specific Fe site. The results provide the basis for a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific [4Fe–4S] cluster chemistry. A similar mechanism is proposed for direct [4Fe–4S]-mediated cleavage of the CoM–S–S–CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.     

Retardation of the G2-M phase progression on gene disruption of RNA binding protein Sam68 in the DT40 cell line

Genes encoding the NarG and NarH subunits of the molybdo-iron-sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe-4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe-4S] cluster and three [4Fe-4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane-bound nitrate reductases whose homologs are found in the bacterial domain.     

Single turnover EPR studies of benzoyl-CoA reductase.

Benzoyl-CoA reductase (BCR) catalyzes the ATP-driven transport of two electrons from a reduced 2[4Fe-4S] ferredoxin to the aromatic ring of benzoyl-CoA. A mechanism involving radical species and very low potential electrons similar to the Birch reduction of aromatics has been suggested for this reaction. The redox centers of BCR have previously been identified, by EPR- and M?ssbauer spectroscopy, to be three cysteine-ligated [4Fe-4S] clusters [Boll et al. (2000) J. Biol. Chem. 275, 31857-31868] with redox potentials more negative than -500 mV. In this work, the catalytic cycle of BCR was studied by freeze-quench experiments; the dithionite reduced enzyme was rapidly mixed with equimolar amounts of benzoyl-CoA and excess MgATP plus dithionite, and subjected to EPR spectroscopic analysis. The turnover period of the enzyme under the conditions used was 3 s. The total S = (1)/(2) spin concentration increased 3-fold very rapidly (within approximately 25 ms). In the course of a single turnover the extent of enzyme reduction decreased again, finally reaching the starting value. An increased magnetic interaction of [4Fe-4S] clusters and the rise of an S = (7)/(2) high-spin EPR signal occurred as second simultaneous and transient events (at approximately 200 ms). Previous work showed that binding of the nucleotide affects the magnetic interaction of [4Fe-4S] clusters, whereas hydrolysis of MgATP is required for the switch to high-spin EPR signals. Finally, two novel transient EPR signals with an isotropic line-shape developed maximally in the late phase of the catalytic cycle ( approximately 1-2 s). These signals differed from those of typical free radicals by shifted g values at g = 2.015 and g = 2.033 and by an unusually fast relaxation rate, suggesting an interaction of these paramagnetic species with [4Fe-4S](+1) clusters. On the basis of these results, we present a proposal for a catalytic cycle involving radical species.     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.     

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.     

Interactions between the molybdenum cofactor and iron-sulfur clusters of Escherichia coli dimethylsulfoxide reductase

We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups of Escherichia coli Me2SO reductase (DmsABC). In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = -120 mV [4Fe-4S] cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA. This interaction is significantly modified in a DmsA-C38S mutant that contains a [3Fe-4S] cluster in DmsA, suggesting that the [3Fe-4S] cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = -120 mV cluster of DmsB. In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway. In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized [3Fe-4S] cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum. In a double mutant, DmsAS176ABC102SC, which contains an engineered [3Fe-4S] cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized [3Fe-4S] cluster and the Mo(V). These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the [4Fe-4S] clusters of DmsB to the Mo-bisMGD of DmsA.     

Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an alpha(3)beta(3) composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K(m) values for perchlorate and chlorate were 27 and <5 microM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed.     

Low-potential iron-sulfur centers in photosystem I: an X-ray absorption spectroscopy study

We have measured the X-ray absorption spectra of Fe in photosystem I (PS I) preparations from spinach and a thermophilic cyanobacterium, Synechococcus sp., to characterize structures of the Fe complexes that function as electron acceptors in PS I. These acceptors include centers A and B, which are probably typical [4Fe-4S] ferredoxins, and X. The structure of X is not known, but its electron paramagnetic resonance (EPR) spectrum has generated the suggestions that it is either a [2Fe-2S] or [4Fe-4S] ferredoxin or an Fe-quinone species. The iron X-ray absorption K-edge and iron extended X-ray absorption fine structure (EXAFS) spectra reveal that essentially all of the 11-14 Fe atoms present in the reaction center are present in the form of Fe-S centers and that not more than 1 atom out of 12 could be octahedral or oxygen-coordinated Fe. This suggests that, besides A and B, additional Fe-S clusters are present which are likely to be X. Our EXAFS spectra cannot be simulated adequately by a mixture of [4Fe-4S] ferredoxins with typical bond lengths and disorder parameters because the amplitude of Fe backscattering is small; however, excellent simulations of the data are consistent with a mixture of [2Fe-2S] ferredoxins and [4Fe-4S] ferredoxins, or with unusually distorted [4Fe-4S] clusters. We presume that the [2Fe-2S] or distorted [4Fe-4S] centers are X. The X-ray absorption spectra of PS I preparations from Synechococcus and spinach are essentially indistinguishable.