The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

The orientation of the magnetic axes of the membrane-bound iron-sulfur clusters of spinach chloroplasts

Spinach chloroplast membranes were oriented onto mylar sheets by partial dehydration, and the orientation of the magnetic axes of membrane-bound paramagnetic clusters determined by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the reduced Rieske iron-sulfur cluster signal is of orthorhombic symmetry oriented with th gy = 1.90 axis orthogonal to the membrane plane and with the gz = 2.03 axis in the membrane plane; the gx-axis is undetectable, presumably due to its broadness. If the Rieske center is a two-iron iron-sulfur cluster, we conclude that the iron-iron axis lies in the plane of the membrane. Illumination reduces the two bound chloroplast iron-sulfur proteins known as Clusters A and B. Center A is oriented such that gx = 1.86 and gy = 1.94 lie at an angle of about 40, and gz = 2.05 is at approximately 25, to the membrane plane. There are two possible orientations of Cluster B depending on the set of g-values assigned to this cluster. For one set of g-values, gz = 2.04 and gx = 1.89 are oriented in the plane of the membrane while gy = 1.92 is orthogonal to the plane. Alternatively, gz = 2.07 and gy = 1.94 are oriented approximately 50 and 40 to the membrane plane respectively, and gx = 1.80 is in the plane of the membrane. An additional light-induced signal at g = 2.15 oriented orthogonal to the plane is currently unexplained, as are other membrane perpendicular signals seen at g = 2.3 and g = 1.73 in dark-adapted samples.     

M?ssbauer spectroscopic studies of the nature of centre X of photosystem I reaction centres from the cyanobacterium Chlorogloea fritschii

Reduced photosystem I samples, which give the electron paramagnetic resonance (EPR) signals associated with A, A and B, and A, B and X centres, have been studied using M?ssbauer spectroscopy. The M?ssbauer spectra obtained from each type of sample is different, which indicates that iron is associated with all three centres. The spectra are similar to those obtained from ferredoxins with 4Fe-4S centres and were fitted with oxidized and reduced components, the relative proportions depending on the degree of reduction of the sample as monitored by EPR. The sample which gave only the A EPR signal showed about 26% of the reduced component, the sample which gave A and B EPR signals showed about 48% of the reduced component, while the sample which gave A, B and X EPR signals showed about 65% of the reduced component. The measurements are consistent with X being a 4Fe-S4 centre.     

Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants

Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 10(5) Pa of H2 at pH 7 giving rise to two distinct S1/2 electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependence of these two [4Fe-4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton-translocating machinery.     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus: a comparative study on the redox properties of the metal clusters present in the dinitrogenase components.

The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of -195 mV (appearance) and -30 mV (disappearance), (2) an axial signal (g(parallel) = 2.00, g perpendicular = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the 'low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the 'high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exhibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: -80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this 'turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.     

Photosynthetic reaction center of green sulfur bacteria studied by EPR

Membrane preparations of two species of the green sulfur bacteria Chlorobium have been studied by EPR. Three signals were detected which were attributed to iron-sulfur centers acting as electron acceptors in the photosynthetic reaction center. (1) A signal from a center designated FB, (gz = 2.07, gy = 1.91, gx = 1.86) was photoinduced at 4 K. (2) A similar signal, FA (gz = 2.05, gy = 1.94, gx = 1.88), was photoinduced in addition to the FB signal upon a short period of illumination at 200 K. (3) Further illumination at 200 K resulted in the appearance of a broad feature at g = 1.78. This is attributed to the gx component of an iron-sulfur center designated FX. The designations of these signals as FB, FA, and FX are based on their spectroscopic similarities to signals in photosystem I (PS I). The orientation dependence of these EPR signals in ordered Chlorobium membrane multilayers is remarkably similar to that of their PS I homologues. A magnetic interaction between the reduced forms of FB and FA occurs, which is also very similar to that seen in PS I. However, in contrast to the situation in PS I, FA and FB cannot be chemically reduced by sodium dithionite at pH 11. This indicates redox potentials for FA and FB which are lower by at least 150 mV than their PS I counterparts. The triplet state of P840, the primary electron donor, could be photoinduced at 4 K in samples which had been preincubated with sodium dithionite and methyl viologen and then preilluminated at 200 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the iron-sulfur cluster N7 (N1c) in the subunit NuoG of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX(27)C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated Delta N1b, Delta N1c, Delta N4, and Delta N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g(x,y,z) = 1.89, 1.94, and 2.06, and g(x,y,z) = 1.91, 1.94, and 2.05 at 6-20 K in wild type, Delta N1b, and Delta N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in Delta N4 and Delta N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knock-out strain, the implicated structural importance of the iron-sulfur binding domains is discussed.     

Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP-inhibited turnover of nitrogenase

Two novel electron paramagnetic resonance (EPR) signals arising from the [1Mo-7Fe-9S-homocitrate] (FeMoco) centres of MoFe protein of Klebsiella pneumoniae nitrogenase (Kp1) were observed following turnover under MgATP-limited conditions. The combination of the nitrogenase Fe protein of Clostridium pasteurianum showed similar signals. The accumulation of MgADP under these conditions causes the normal EPR signal of dithionite-reduced Kp1 (with g=4.3, 3.6, 2.01) to be slowly converted to novel signals with g=4.74, 3.32, 2.00 and g=4.58, 3.50, 1.99. These signals do not form in incubation of protein mixtures containing only MgADP, thus they may be associated with trapped intermediates of the catalytic cycle.     

M?ssbauer studies of Escherichia coli sulfite reductase complexes with carbon monoxide and cyanide. Exchange coupling and intrinsic properties of the [4Fe-4S] cluster

M?ssbauer studies of the hemoprotein subunit (SiR) of E. coli sulfite reductase have shown that the siroheme and the [4Fe-4S] cluster are exchange-coupled. Here we report M?ssbauer studies of SiR complexed with either CO or CN- and of SiR in the presence of the chaotropic agent dimethyl sulfoxide (Me2SO). The spectra of one-electron-reduced SiR X CN show that all five iron atoms reside in a diamagnetic environment; the ferroheme X CN complex is low spin and the [4Fe-4S] cluster is in the 2+ oxidation state. Titration with ferricyanide affords a CN- complex of oxidized SiR in which the siroheme iron is low spin ferric, with the cluster remaining in the 2+ state. At low temperatures, paramagnetic hyperfine interactions are observed for the iron sites of the cluster, suggesting that it is exchange-coupled to the heme iron. Reduction of one-electron-reduced SiR X CN and SiR X CO yields complexes with "g = 1.94"-type EPR signals showing that the second electron is accommodated by the iron-sulfur cluster. The fully reduced complexes yield well resolved M?ssbauer spectra which were analyzed in the spin Hamiltonian formalism. The analysis shows that the cluster subsites are equivalent in pairs, one pair having properties reminiscent of ferric sites whereas the other pair has features more typical of ferrous sites. The M?ssbauer spectra of oxidized SiR kept in 60% (v/v) Me2SO are virtually identical with those observed for SiR in standard buffer, implying that the coupling is maintained in the presence of the chaotrope. Fully reduced SiR displays an EPR signal with g values of g = 2.53, 2.29, and 2.07. In 60% Me2SO, this signal vanishes and a g = 1.94 signal develops; this transition is accompanied by a change in the spin state of the heme iron from S = 1 (or 2) to S = O.     

Characterization of iron-sulphur centres of plant mitochondria by microwave power saturation.

The electron spin relaxation of iron-sulphur centres and ubisemiquinones of plant mitochondria was studied by microwave power saturation of the respective EPR signals. In the microwave power saturation technique, the experimental saturation data were fitted by a least-squares procedure to a saturation function which is characterized by the power for half-saturation (P1/2) and the inhomogeneity parameter (b). Since the theoretical saturation curves were based on a one-electron spin system, it became possible to differentiate between EPR signals of iron-sulphur centres which have similar g values but different P1/2 values. If the difference in the P1/2 values of the overlapped components was small, no significant deviation from these theoretical saturation curves was observed, as shown for the overlapped signals of centre S-3 and the Ruzicka centre of mung bean mitochondria. By contrast, the microwave power saturation data for the g = 1.93 signal (17--26 K) of Arum maculatum submitochondrial particles reduced by succinate could not be fitted using one-electron saturation curves. Reduction by NADH resulted in a stronger deviation. Since the iron-sulphur centres of Complex I were present only in an unusually low concentration in A. maculatum mitochondria, it was proposed that an iron-sulphur centre of the external NADH dehydrogenase contributes to the spectrum of centre S-1. For mung bean mitochondria, the g = 1.93 signal below 20 K could be attributed mainly to centre N-2. The microwave power saturation technique was also suitable for detecting magnetic interactions between paramagnetic centres. From the saturation data of the complex spectrum attributable to centre S-3 and an interacting ubisemiquinone pair in mung bean mitochondria (oxidized state) followed that centre S-3 has a faster electron spin relaxation than the ubisemiquinone molecules. It is noteworthy that the differences in the relaxation rates were maintained despite the interaction between centre S-3 and the ubisemiquinones. Furthermore, a relaxation enhancement was observed for centre S-1 of A. maculatum submitochondrial particles upon reduction of centre S-2 by dithionite. This indicated a magnetic interaction between centres S-1 and S-2.     

Comparison of the EPR properties of photosystem I iron-sulphur centres A and B in spinach and barley

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.     

M?ssbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2

We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with M?ssbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits M?ssbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the M?ssbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and M?ssbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.     

Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas.

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.     

M?ssbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum

We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA. As shown here, this protein contains a [4Fe-4S]1+/2+ cluster in addition to a corrinoid. The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand. Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein. The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state. Optical studies suggest that the Co3+-CH3 corrinoid is also base-off. In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of [4Fe-4S]2+ clusters. The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin. The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86. The M?ssbauer data show that these signals result from [4Fe-4S]1+ clusters. Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single [4Fe-4S]1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state. Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and M?ssbauer spectra of typical [4Fe-4S]1+ clusters. A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P. (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al. (1973).     

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

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

The bound electron acceptors in green sulfur bacteria: resolution of the g-tensor for the F(X) iron-sulfur cluster in Chlorobium tepidum

The photosynthetic reaction center (RC) of green sulfur bacteria contains two [4Fe-4S] clusters named F(A) and F(B), by analogy with photosystem I (PS I). PS I also contains an interpolypeptide [4Fe-4S] cluster named F(X); however, spectroscopic evidence for an analogous iron-sulfur cluster in green sulfur bacteria remains equivocal. To minimize oxidative damage to the iron-sulfur clusters, we studied the sensitivity of F(A) and F(B) to molecular oxygen in whole cells of Chlorobium vibrioforme and Chlorobium tepidum and obtained highly photoactive membranes and RCs from Cb. tepidum by adjusting isolation conditions to maximize the amplitude of the F(A)(-)/F(B)(-) electron paramagnetic resonance signal at g = 1.89 (measured at 126 mW of microwave power and 14 K) relative to the P840(+) signal at g = 2.0028 (measured at 800 microW of microwave power and 14 K). In these optimized preparations we were able to differentiate F(X)(-) from F(A)(-)/F(B)(-) by their different relaxation properties. At temperatures between 4 and 9 K, isolated membranes and RCs of Cb. tepidum show a broad peak at g = 2.12 and a prominent high-field trough at g = 1.76 (measured at 126 mW of microwave power). The complete g-tensor of F(X)(-), extracted by numerical simulation, yields principal values of 2.17, 1.92, and 1. 77 and is similar to F(X) in PS I. An important difference from PS I is that because the bound cytochrome is available as a fast electron donor in Chlorobium, it is not necessary to prereduce F(A) and F(B) to photoaccumulate F(X)(-).     

Study of the respiratory chain in Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy

Low-temperature electron spin resonance spectroscopy was used to investigate the redox centres of Micrococcus luteus membranes. Three different types of iron-sulphur centres were distinguished. Two of these, a [4Fe-4S]3+-type cluster giving rise to a signal at g = 2.01 in the oxidized state and a [2Fe-2S] cluster with a spectrum at g = 2.03 and 1.93 in the reduced state, were attributable to succinate dehydrogenase. Another, generating signals in the reduced state at g = 2.027, 1.90 and 1.78 was identified as a 'Rieske' iron-sulphur centre. This latter cluster had a mid-point potential (pH 7.0) of +130 mV. In addition, signals characteristic of high-spin ferric haem (g = 6.20), low-spin ferric haem (g = 3.67, 3.36 and 3.01) and Cu2+ (g = 2.18 and 2.02) were also detected. The ferric-haem features, together with the Cu2+ and 'Rieske' centres, were enriched in membrane residues insoluble in Triton X-100, which are known from difference spectroscopy to contain cytochromes b-560, c-550 and a-601 (aa3 oxidase). The signals demonstrated by electron spin resonance for M. luteus membranes showed marked similarities to those documented for the complexes II, III, and IV of mitochondria. However, signals analogous to complex I (NADH-ubiquinone reductase) could not be demonstrated for M. luteus membranes.     

Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)     

Paramagnetic centers of carbon monoxide dehydrogenase from aceticlastic Methanosarcina barkeri

Carbon monoxide dehydrogenase from Methanosarcina barkeri, purified to 95% homogeneity, contains 30 Fe, 2 Ni, 1 Zn, and 1 Cu (per alpha 2 beta 2 enzyme). Core extrusion experiments indicate 6 [4Fe-4S] clusters/tetramer, and electron paramagnetic resonance (epr) spectroscopy detects at least one of these clusters, in the reduced form, with apparent g values of 2.05, 1.94, and 1.90, and Em9.2-390 mV. A second epr signal, also seen in the reduced enzyme, has apparent g values of 2.005, 1.91, and 1.76, and Em9.2-35 mV. Two signals were seen in thionin-oxidized enzyme, one with a line shape suggestive of Cu(II), and the other resembling that of a [3Fe-4S] cluster. The enzymes nonphysiological substrate, CO, caused several spectral changes to the reduced enzyme, most notably a shift of the g = 1.76 feature to g = 1.73.