Spectroscopic evidence for a [3Fe-4S] cluster in spinach glutamate synthase.

The combination of low temperature EPR, magnetic circular dichroism, and resonance Raman spectroscopies reveals the presence of a single [3Fe-4S]+,0 center as the sole iron-sulfur prosthetic group in glutamate synthase from spinach leaves. The electronic, magnetic, and structural properties of the oxidized and reduced cluster are analogous with those of similar clusters in bacterial ferredoxins. It was not possible to convert the [3Fe-4S] cluster to a [4Fe-4S] cluster by incubating with iron under reducing conditions. Taken together with the published amino acid sequence data for plant and bacterial glutamate synthases, this suggests that the [3Fe-4S] cluster is not an isolation artifact resulting from oxidative degradation of a [4Fe-4S] cluster. The likelihood that a [3Fe-4S] cluster is an intrinsic component of all plant and bacterial glutamate synthases is discussed.     

Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.     

Glutathione-complexed [2Fe-2S] clusters function in Fe–S cluster storage and trafficking

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS₄) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe–S cluster transfer reactions. UV–vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS₄) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS₄) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe–S cluster proteins, thereby supporting a possible physiological role for such centers.     

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.     

Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy

Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.     

Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies.

Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.     

The high potential iron-sulfur center in Escherichia coli fumarate reductase is a three-iron cluster

The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases.     

Reactivity of nitric oxide with the [4Fe-4S] cluster of dihydroxyacid dehydratase from Escherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.     

Evidence for the existence of [2Fe-2S] as well as [4Fe-4S] clusters among FA,FB and FX. Implications for the structure of the Photosystem I reaction center

Core extrusion of the bound iron-sulfur centers from spinach Photosystem I showed the presence of [2Fe-2S] clusters as well as [4Fe-4S] clusters among F_A, F_B and F_X. Extrusion of the iron-sulfur ensemble was not quantitative; however, the presence of [2Fe-2S] clusters correlated with higher concentration of unfolding solvent. Since F_X is highly resistant to denaturation, and since F_A and F_B are known to contain [4Fe-4S] clusters, the [2Fe-2S] clusters are assigned to F_X. The presence of [2Fe-2S] clusters in Photosystem I has significance in the structure and organization of F_X on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a Photosystem I subunit may consist of two approx. 64-kDa proteins bridged by a single [2Fe-2S] cluster. The complete reaction center would consist of two subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting F_X.     

Thermodynamic redox properties governing the half-reduction characteristics of histamine dehydrogenase from Nocardioides simplex

Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers' redox potential.     

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.     

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.     

Iron-sulphur clusters with labile metal ions.

A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd III from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [formula: see text] The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]0 cluster for divalent ions lie in the sequence Cd greater than Zn much greater than Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S]1+ as well as for [3Fe-4S]0. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-mu 2-sulphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least one additional ligand which may be from solvent (H2O or OH-) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn(II), that may trigger conformational change.     

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]²⁺ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]²⁺ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]²⁺ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]²⁺ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]²⁺ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.     

Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.     

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.     

Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron-sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). In this work, the ground- and excited-state electronic properties of the paramagnetic Fe-S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable-temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe-4S](+) clusters in dithionite-reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe-4S](3+) clusters in oxidized HDR samples treated separately with the CoM-SH and CoB-SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.