Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.     

The iron-sulfur cluster composition of Escherichia coli nitrate reductase

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.     

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.     

The prosthetic groups in succinate dehydrogenase. Number and stoichiometry

I. Succinate:Q oxidoreductase (EC 1.3.99.1) as present in beef-heart submitochondrial particles contains equal amounts of FAD, a [2Fe-2S] cluster and a [4Fe-4S] cluster. Both Fe-S clusters are reducible by succinate. 2. A second type of [2Fe-2S] cluster, called center S-2, that has been proposed to be present in purified preparations of succinate dehydrogenase and isolated Complex II (Ohnishi, T., Winter, D.B., Lim, J. and King, T.E. (1973) Biochem. Biophys. Res. Commun. 53, 231--237) is an artifact introduced by the purification procedure. 3. It is suggested that the 70 000 dalton subunit which is known to bind the flavin, accomodates also the [4Fe-4S] cluster whereas the 28 000 dalton subunit contains the [2Fe-2S] cluster.     

The iron-sulfur centers of the soluble [NiFeSe] hydrogenase, from Desulfovibrio baculatus (DSM 1743). EPR and M?ssbauer characterization

The soluble (cytoplasmic plus periplasmic) Ni/Fe-S/Se-containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe-enriched medium, and its iron-sulfur centers were extensively characterized by M?ssbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe-4S] center, either in the native (as isolated) or in the hydrogen-reduced states. In the native state, the non-heme iron atoms are arranged as two diamagnetic [4Fe-4S]2+ centers. Upon reduction, these two centers exhibit distinct and unusual M?ssbauer spectroscopic parameters. The centers were found to have similar mid-point potentials (approximately -315 mV) as determined by oxidation-reduction titratins followed by EPR.     

Electron paramagnetic resonance studies of mammalian succinate dehydrogenase. Detection of the tetranuclear cluster S2

Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.     

Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase

Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtilis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.     

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 signals of NADH: Q oxidoreductase. Shape and intensity.

(1) The EPR spectrum of Center 1 of NADH dehydrogenase in isolated Complex I or submitochondrial particles from beef heart consists of two overlapping nearly axial signals of the same intensity. They are defined as Center 1a (gll = 0.021, gl = 1.938) and Center 1b (gll = 2.021, gl = 1.928). (2) The line shape of the EPR spectrum of the Center 3+4 can be interpreted as an overlap of two rhombic signals of the same intensity. We define Center 3 by the g-values: gz=2.103, gy = 1.93-1.94, gx=1.884, and Center 4 by the values gz=2.04, gy=1.92-1.93, gx=1.863. (3) Direct quantitation of the individuals signals as well as computer stimulation suggests that the amount of the Centers 1a and 1b is only 25% of that of the other individuals centers and FMN. As EPR spectra of beef-heart submitochondrial particles at 10-20 K are nearly identical to those of Complex I, the same relative concentrations of the Fe-S centers are also present in the particles. (4) The signals either observed by us in EPR spectra of Complex I and submitochondrial particles at 4.2 K and high microwave powers can now be explained without assuming more than 5 paramagnetic centers in NADH dehydrogenase.     

M?ssbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5

The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides. We have studied oxidized samples of the enzyme with M?ssbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01. The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances. One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz. These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The M?ssbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms. Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated [4Fe-4S] cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a [4Fe-4S] core. The M?ssbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type [4Fe-4S] clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.     

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.     

An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5

Previous M?ssbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel M?ssbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.     

Two modes of irreversible inactivation of the mitochondrial electron-transfer system by tetradecanoic acid

Bovine heart submitochondrial particles were incubated for 2-6 h at 37 degrees C with various concentrations of tetradecanoic acid, and the effects on the activities, the total acid-labile sulphide content and EPR spectra of the electron transfer system were studied. Two distinct time-dependent processes of the slow irreversible inactivation of the electron-transfer system were found. They differ in the concentration of tetradecanoic acid required. The more specific effect, induced by 100-400 nmol tetradecanoic acid per mg protein, consists of a selective blockage of electron transfer between the Fe-S clusters of the NADH dehydrogenase and ubiquinone, without damage to any of the Fe-S clusters. Higher concentrations of tetradecanoic acid caused gradual destruction of all Fe-S clusters of NADH dehydrogenase and of the 3-Fe cluster of succinate dehydrogenase, leading to complete inactivation of both NADH and succinate oxidation.     

Identification of FX in the heliobacterial reaction center as a [4Fe-4S] cluster with an S = 3/2 ground spin state

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The M?ssbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.     

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)     

57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and M?ssbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in M?ssbauer spectroscopy. ENDOR studies confirm the M?ssbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)     

Formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774: isolation and spectroscopic characterization of the active sites (heme, iron-sulfur centers and molybdenum)

An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO₂, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150?kDa (three different subunits: 88, 29 and 16?kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S]^2+/1+ centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g _max=2.050, g _med=1.947, g _min=1.896) and an [Fe-S] center II (g _max=2.071, g _med=1.926, g _min=1.865). Mössbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S]^2+/1+ centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.     

3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.     

Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin. Possible new fingerprints for three iron clusters

We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus. First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers. This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2. Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the [4Fe-4S]+ (S = 1/2) and the reduced 3Fe center. A theoretical explanation is given for the observation of interaction signals with constant effective g values.     

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.