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.     

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.     

The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state

We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). FS0 is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures (<15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = (3)/(2) ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = (1)/(2) and DeltaS = (3)/(2) transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.     

Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)     

The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits

The hndABCD operon from Desulfovibrio fructosovorans encodes an uncommon heterotetrameric NADP-reducing iron hydrogenase. The presence of a [2Fe-2S] cluster likely located in the C-terminal region of the HndA subunit has already been revealed. We have cloned and expressed the truncated hndA gene in Escherichia coli to isolate the structural [2Fe-2S] module. Optical and EPR spectra are found identical to that of the native HndA subunit and the midpoint redox potential (-385 mV) is similar to that of the native protein (-395 mV). These results clearly demonstrate that the C-terminal region of HndA is a structurally independent [2Fe2S] ferredoxin-like domain. In the same way, the N-terminal domain of the HndD subunit was overproduced in E. coli and characterized. The presence of a [2Fe-2S] cluster was evidenced by optical spectroscopy. The midpoint redox potential (-380 mV) of this domain was found very close to that of the truncated HndA subunit but the EPR properties were significantly different. The various EPR properties allowed us to observe an electron exchange between the two [2Fe-2S] ferredoxin-like domains of the HndA and HndD subunits. Moreover, domain-domain interactions, observed by far-western experiments, indicate that these subunits are direct partners in the native complex.     

Formation and properties of [4Fe-4S] clusters on the IscU scaffold protein

Rapid and quantitative reductive coupling of two [2Fe-2S]2+ clusters to form a single [4Fe-4S]2+ cluster on the homodimeric IscU Fe-S cluster scaffold protein has been demonstrated by UV-visible absorption, M?ssbauer, and resonance Raman spectroscopies, using dithionite as the electron donor. Partial reductive coupling was also observed using reduced Isc ferredoxin, which raises the possibility that Isc ferredoxin is the physiological reductant. The results suggest that reductive coupling of adjacent [2Fe-2S]2+ clusters assembled on IscU provides a general mechanism for the final step in the biosynthesis of [4Fe-4S]2+ clusters. The [4Fe-4S]2+ center on IscU can be reduced to a S = 1/2[4Fe-4S]+ cluster (g parallel = 2.06 and g perpendicular = 1.92), but the low midpoint potential (< -570 mV) and instability of the reduced cluster argue against any physiological relevance for the reduced cluster. On exposure to O2, the [4Fe-4S]2+ cluster on IscU degrades via a semistable [2Fe-2S]2+ cluster with properties analogous to those of the [2Fe-2S]2+ center in [2Fe-2S]2+ IscU. It is suggested that the ability of IscU to accommodate either [2Fe-2S]2+ or [4Fe-4S]2+ clusters in response to cellular redox status and/or oxygen levels may provide an effective way to populate appropriately cluster-loaded forms of IscU for maturation of different types of [Fe-S] proteins.     

An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase

Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters.     

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties.In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH?8 by 230?mV to 400?mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.     

Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and M?ssbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.     

Pyrococcus furiosus glyceraldehyde 3-phosphate oxidoreductase has comparable W(6+/5+) and W(5+/4+) reduction potentials and unusual [4Fe-4S] EPR properties

Pyrococcus furiosus glyceraldehyde 3-phosphate oxidoreductase has been characterized using EPR-monitored redox titrations. Two different W signals were found. W(1)(5+) is an intermediate species in the catalytic cycle, with the midpoint potentials E(m)(W(6+/5+))=-507 mV and E(m)(W(5+/4+))=-491 mV. W(2)(5+) represents an inactivated species with E(m)(W(6+/5+))=-329 mV. The cubane cluster exhibits both S=3/2 and S=1/2 signals with the same midpoint potential: E(m)([4Fe-4S](2+/1+))=-335 mV. The S=1/2 EPR signal is unusual with all g values below 2.0. The titration results combined with catalytic voltammetry data are consistent with electron transfer from glyceraldehyde 3-phosphate first to the tungsten center, then to the cubane cluster and finally to the ferredoxin.     

Oxidation-reduction properties of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Native Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase contains a [4Fe-4S] cluster in the diamagnetic (+2) state. The cluster is essential for catalytic function, even though amidotransferase does not catalyze a redox reaction. The ability of the Fe-S cluster to undergo oxidation and reduction reactions and the consequences of changes in the redox state of the cluster for enzyme activity were studied. Treatment of the enzyme with oxidants resulted in either no reaction or complete dissolution of the Fe-S cluster and loss of activity. A stable +3 oxidation state was not detected. A small amount of paramagnetic species, probably an oxidized 3Fe cluster, was formed transiently during oxidation. The native cluster was poorly reduced by dithionite, but it could be readily reduced to the +1 state by photoreduction with 5-deazaflavin and oxalate. The reduced enzyme did not display an EPR spectrum typical of [4Fe-4S] ferredoxins in the +1 state, unless it was prepared under denaturing conditions. M?ssbauer spectroscopy of reduced 57Fe-enriched amidotransferase confirmed that the cluster was in the +1 state, but the magnetic properties of the reduced cluster observed at 4.2 K indicated that it is characterized by a ground state spin S greater than or equal to 3/2. The midpoint potential of the +1/+2 couple was too low to measure accurately by conventional techniques, but it was below -600 mV, which is 100 mV more negative than reported for [4Fe-4S] clusters in bacterial ferredoxins. Fully reduced amidotransferase had about 40% of the activity of the native enzyme in glutamine-dependent phosphoribosylamine formation. The fact that both the +1 and +2 forms of the enzyme are active indicates that the cluster does not function as a site of reversible electron transfer during catalysis.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.     

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.     

Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.     

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.     

The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.     

Adenosine triphosphate-induced electron transfer in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans catalyzes the chemical difficult elimination of water from (R)-2-hydroxyglutaryl-CoA to glutaconyl-CoA. The enzyme consists of two oxygen-sensitive protein components, the homodimeric activator (A) with one [4Fe-4S]1+/2+ cluster and the heterodimeric dehydratase (D) with one nonreducible [4Fe-4S]2+ cluster and reduced riboflavin 5'-monophosphate (FMNH2). For activation, ATP, Mg2+, and a reduced flavodoxin (16 kDa) purified from A. fermentans are required. The [4Fe-4S](1+/2+) cluster of component A is exposed to the solvent since it is accessible to iron chelators. Upon exchange of the bound ADP by ATP, the chelation rate is 8-fold enhanced, indicating a large conformational change. Oxidized component A exhibits ATPase activity of 6 s(-1), which is completely abolished upon reduction by one electron. UV-visible spectroscopy revealed a spontaneous one-electron transfer from flavodoxin hydroquinone (E(0)' = -430 mV) to oxidized component A, whereby the [4Fe-4S]2+ cluster of component A became reduced. Combined kinetic, EPR, and M?ssbauer spectrocopic investigations exhibited an ATP-dependent oxidation of component A by component D. Whereas the [4Fe-4S]2+ cluster of component D remained in the oxidized state, a new EPR signal became visible attributed to a d1-metal species, probably Mo(V). Metal analysis with neutron activation and atomic absorption spectroscopy gave 0.07-0.2 Mo per component D. In summary, the data suggest that in the presence of ATP one electron is transferred from flavodoxin hydroquinone via the [4Fe-4S]1+/2+ cluster of component A to Mo(VI) of component D, which is thereby reduced to Mo(V). The latter may supply the electron necessary for transient charge reversal in the unusual dehydration.     

Investigation of the environment surrounding iron-sulfur cluster 4 of Escherichia coli dimethylsulfoxide reductase

Iron-sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E(m) values) play a major role in defining the rate at which electrons are shuttled. The E(m) values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1-FS4) with E(m) values between -50 and -330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH(2)) binding, and protein control of its E(m). Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB-DmsC interface and are critical for the binding of the MQH(2) inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH(2) to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin-spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4([3Fe)(-)(4S])). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E(m) of FS4([3Fe)(-)(4S]) from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4([3Fe)(-)(4S]) lower its E(m), but mutations of negatively charged residues have negligible effects. The E(m) of FS4([3Fe)(-)(4S]) in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4([3Fe)(-)(4S]) E(m) of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E(m) for FS4([3Fe)(-)(4S]) of DmsB is approximately 275 mV.     

Unusual NMR, EPR, and M?ssbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and M?ssbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its M?ssbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.     

Multi-frequency EPR and high-resolution M?ssbauer spectroscopy of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). Characterization of a supercluster and superspin model protein.

The putative [6Fe-6S] prismane cluster in the 6-Fe/S-containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in 57Fe, and has been characterized in detail by S-, X-, P- and Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution 57Fe M?ssbauer spectroscopy. In EPR-monitored redox-equilibrium titrations, the cluster is found to be capable of three one-electron transitions with midpoint potentials at pH 7.5 of +285, +5 and -165 mV. As the fully reduced protein is assumed to carry the [6Fe-6S]3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments: [6Fe-6S]3+, [6Fe-6S]4+, [6Fe-6S]5+, [6Fe-6S]6+. The fully oxidized 6+ state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electron-reduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is low-spin S = 1/2; the remainder has an unusually high spin S = 9/2. The S = 1/2 EPR spectrum is significantly broadened by ligand (2.3 mT) and 57Fe (3.0 mT) hyperfine interaction, consistent with a delocalization of the unpaired electron over 6Fe and indicative of at least some nitrogen ligation. At 35 GHz, the g tensor is determined as 1.971, 1.951 and 1.898. EPR signals from the S = 9/2 multiplet have their maximal amplitude at a temperature of 12 K due to the axial zero-field splitting being negative, D approximately -0.86 cm-1. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of [E/D] = 0.061. A second component has g = 9.7, 8.1 and 6.65 and [E/D] = 0.108. When the protein is reduced to the 4+ intermediate state, the cluster is silent in normal-mode EPR. An asymmetric feature with effective g approximately 16 is observed in parallel-mode EPR from an integer spin system with, presumably, S = 4. The fully reduced 3+ state consists of a mixture of two S = 1/2 ground state. The g tensor of the major component is 2.010, 1.825 and 1.32; the minor component has g = 1.941 and 1.79, with the third value undetermined. The sharp line at g = 2.010 exhibits significant convoluted hyperfine broadening from ligands (2.1 mT) and from 57Fe (4.6 mT). Zero-field high-temperature M?ssbauer spectra of the protein, isolated in the 5+ state, quantitatively account for the 0.8 fractional enrichment in 57Fe, as determined with inductively coupled plasma mass spectrometry.(ABSTRACT TRUNCATED AT 400 WORDS)