EPR-active Species Research Articles

X-ray absorption and resonance Raman studies of methyl-coenzyme M reductase indicating that ligand exchange and macrocycle reduction accompany reductive activation.

Methyl-coenzyme M reductase (MCR) catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). MCR contains a nickel hydrocorphin cofactor at its active site, called cofactor F(430). Here we present evidence that the macrocyclic ligand participates in the redox chemistry involved in catalysis. The active form of MCR, the red1 state, is generated by reducing another spectroscopically distinct form called ox1 with titanium(III) citrate. Previous electron paramagnetic resonance (EPR) and (14)N electron nuclear double resonance (ENDOR) studies indicate that both the ox1 and red1 states are best described as formally Ni(I) species on the basis of the character of the orbital containing the spin in the two EPR-active species. Herein, X-ray absorption spectroscopic (XAS) and resonance Raman (RR) studies are reported for the inactive (EPR-silent) forms and the red1 and ox1 states of MCR. RR spectra are also reported for isolated cofactor F(430) in the reduced, resting, and oxidized states; selected RR data are reported for the (15)N and (64)Ni isotopomers of the cofactor, both in the intact enzyme and in solution. Small Ni K-edge energy shifts indicate that minimal electron density changes occur at the Ni center during redox cycling of the enzyme. Titrations with Ti(III) indicate a 3-electron reduction of free cofactor F(430) to generate a stable Ni(I) state and a 2-electron reduction of Ni(I)-ox1 to Ni(I)-red1. Analyses of the XANES and EXAFS data reveal that both the ox1 and red1 forms are best described as hexacoordinate and that the main difference between ox1 and red1 is the absence of an axial thiolate ligand in the red1 state. The RR data indicate that cofactor F(430) undergoes a significant conformational change when it binds to MCR. Furthermore, the vibrational characteristics of the ox1 state and red1 states are significantly different, especially in hydrocorphin ring modes with appreciable C=N stretching character. It is proposed that these differences arise from a 2-electron reduction of the hydrocorphin ring upon conversion to the red1 form. Presumably, the ring-reduction and ligand-exchange reactions reported herein underlie the enhanced activity of MCR(red1), the only form of MCR that can react productively with the methyl group of methyl-SCoM.

Biochemical, Mössbauer, and EPR studies of the diiron cluster of phenol hydroxylase from Pseudomonas sp. strain CF 600.

Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. Mössbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation. In addition, Mössbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.

EPR studies on gamma-irradiated snails hard tissues

Some features of the X-band EPR spectra taken after γ-irradiation of some seasnails and snail ( Helix lukortium) are reported. The estimated EPR parameters ( g 1=2.0055, g 2=2.003, g 3=2.002, g 4=2.000 and g z =1.996) of all samples suggest a complex spectrum consisting of three different EPR active species. The intensity of the EPR response which is stable for more than 1 year exhibits different behaviour with respect to the absorbed dose.

On the iron–sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene.

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

Structure and Physical Properties of Trigonal Monopyramidal Iron(II), Cobalt(II), Nickel(II), and Zinc(II) Complexes

A series of trigonal monopyramidal complexes of the tripodal ligand tris((N-tert-butylcarbamoyl)methyl)aminato, [1But]3-, have been synthesized and characterized. The structures of [Co1But]-, [Zn1But]-, and [Ni1But]- confirm that trigonal monopyramidal coordination geometry occurs in these complexes where the three amidate nitrogens are arranged in the trigonal plane and the amine nitrogen is bonded apically to the metal ions. The solid-state structures of [Co1But]-, [Zn1But]-, and [Ni1But]- are nearly identical indicating that the trigonal ligand [1But]3- enforces the trigonal monopyramidal structure in these metal ions. Crystal data: K[Co1But]·0.5DMF crystallizes in the monoclinic space group C2/c, with cell dimensions a = 18.844(4) Å, b = 9.809(3) Å, c = 28.715(13) Å, β = 102.70°, and Z = 8; (NEt4)[Zn1But]·THF crystallizes in the monoclinic space group P21/c, with cell dimensions a = 13.244(3) Å, b = 11.285(5) Å, c = 25.625(3) Å, β = 104.45(1)°, and Z = 4. The 1H NMR spectrum of the diamagnetic [Zn1But]- also suggests that the complex retains its C3 symmetry in solution. Room-temperature magnetic susceptibility measurements show that [Fe1But]-, [Co1But]-, and [Ni1But]- are high spin. The cyclic voltammetry of [Co1But]- and [Ni1But]- at a glassy carbon surface and at a scan rate of 100 mV s-1 shows quasi-reversible one electron oxidation at E1/2 = 0.77 (ΔEp = 93 mV, ipcipa-1 = 0.69) and 0.56 (ΔEp = 75 mV, ipcipa-1 = 0.79) V versus SCE, respectively. However, at slower scan rates these redox processes become irreversible and attempts to isolate the oxidized products at room temperature were unsuccessful. The chemical oxidation of [Ni1But]- with [Fe(bpy)3]3- in 1:1 propionitrile−DMF mixture at −75 °C generated a EPR-active species (77 K, g1 = 2.29, g2 = 2.16, g3 = 2.03, a3 = 20 G) assigned to a Ni(III) complex with rhombic symmetry. [Fe1But]- shows one irreversible oxidation (Ep,a = 0.05 V versus SCE) under the same conditions. These results are consistent with [1But]3- being able to stabilize trigonal monopyramidal complexes of low-valent metal ions.

Accessibility of 17-Electron Structures for Cyclopentadienylchromium(III) Compounds. 2. Cyanide Derivatives with 15- and 17-Electron Configurations

Ion metathesis of [(ring)CrCl2]2 (ring = Cp, Cp*) with excess KCN in water affords the complexes [(ring)Cr(CN)3]-, which have been isolated as PPh4+ salts and characterized by elemental (C, H) analysis, 1H NMR, magnetic suceptibility, and an X-ray investigation for the Cp* species. The interaction of these complexes with excess CN- generate equilibrium amounts of the EPR-active, 17-electron [(ring)Cr(CN)4]2- adducts. The equilibrium constant for this addition process is 2.3 ± 0.1 M-1 for ring = Cp and (4.4 ± 0.2) × 10-3 M-1 for ring = Cp* at 298 K. Either treatment of [CpCr(CN)3]- with monodentate (L) or bidentate (L2) tertiary phosphine ligands (e.g. PMe3, PMe2Ph, dmpm, dmpe) or reaction between CpCrCl2(η1-L2) or [CpCrCl2L + L] and excess Me3SiCN leads to solutions containing small equilibrium amounts of EPR-active species which are characterized by hyperfine coupling to two equivalent P nuclei and are interpreted as the 17-electron CpCr(CN)2L2. The thermal accessibility of these 17-electron Cr(III) cyan...

An EPR study of as-grown and photoinduced defects in C 60 thin films

EPR active species were found in irradiated C 60 thin films at room temperature both as-grown and after irradiation by light. The samples exhibit a singlet Lorentzian line with g = 2.0026 ± 0.0002 and Δ H pp = 0.124 ± 0.005 mT which may be attributed to holes localized at rapidly rotating C 60 molecules, i.e. C 60 + free radicals. The bulk density of the C 60 + radicals in the as-grown samples depends on the conditions of the sample preparation and reaches 1 spin per 10 000 molecules for fast-grown films. Annealing at 570 K leads to a rapid decrease in that density. Irradiation of the C 60 thin films by visible and UV light leads to a significant increase in the number of the same C 60 + radicals. The total number of photoinduced defects depends on the light intensity and time of exposure. The irradiation by visible light is active only in the presence of oxygen whereas the UV irradiation acts also when the sample is kept in an inert atmosphere. At ambient conditions the C 60 + radicals are metastable with life time of several months.

Spectromagnetic investigation of the active species in the oxidation of propenoidic phenols catalysed by [N,N′-bis(salicylidene)ethane-1,2-diaminato]cobalt(II) *

The oxidation of propenoidic phenols by molecular oxygen, catalysed by [N,N′-bis(salicylidene)ethane-1,2-diaminato]cobalt(II) [Co(salen)], was studied in different solvents and for variously substituted phenols to find processes alternative to those reported for the removal of polyphenols from waste waters in the paper industry. The reaction of methyl E-4-hydroxy-3-methoxycinnamate (E-methyl ferulate) selectively gave the corresponding 4-hydroxybenzoic acid and 4-hydroxybenzaldehyde; yields are highest in chloroform, good in methanol and very low in pyridine. Conversion was high with E-methyl ferulate, lower with methyl E-4-hydroxycinnamate, while the other phenols, methyl E-3-hydroxy-4-methoxycinnamate (E-methyl isoferulate) and methyl E-3-chloro-4-hydroxycinnamate, did not react. An EPR investigation of the reaction mixtures, performed on samples taken at different reaction times, demonstrated that the most probable mechanism involves reactions (i)–(iii). The superoxocobalt radical, [CoIII(salen)(ROH)(O2)]–, and the phenoxy cobalt radical, [CoIII(salen)(RO–)(RO˙)], are the EPR-active species; RO˙ is suggested easily to dissociate from [CoIII(salen)(RO–)(RO˙)] and, reacting with O2 at the β-carbon of the allyl substituent, probably gives a dioxethane. This decomposes to aldehyde and acid. The phenol electron-donor properties promote the formation of the superoxocobalt derivative and consequently favour the formation of the phenoxy cobalt radical; a too high stability of this radical does not favour its evolution into oxidation products.

Electron paramagnetic resonance study of the S = ½ ground state of a radiolysis-generated manganese(III)–trimanganese(IV) form of [MnIV4O6(bipy)6]4+ (bipy = 2,2′-bipyridine). Comparison with the photosynthetic Oxygen Evolving Complex †

gamma-Ray irradiation at liquid nitrogen temperature of a dimethylformamide solution of the tetranuclear complex [(Mn4O6)-O-IV(bipy)(6)](4+) (bipy = 2,2'-bipyridine) allowed the generation of the first mixed-valence tetranuclear system containing Mn-III and Mn-IV ions and exhibiting a S = 1/2 ground state. The X-band EPR spectrum of this tetranuclear system has been obtained. Simulations have been undertaken and the Mn hyperfine coupling tensors determined clearly show a (MnMn3IV)-Mn-III, composition for the EPR active species. A general approach for the analysis of the isotropic components of the Mn hyperfine tensors is presented in detail. This allowed the determination of the spin projection value for each Mn site. A three J coupling scheme assuming that the linear topology of the starting compound remains is able to reproduce these spin projection values if and only if the Mn-III ion is located at a terminal position in a N4O2 environment. The EPR signal of this [Mn4O6(bipy)(6)](3+) species is compared with the multiline signal observed in the S-2 state of the photosynthetic Oxygen Evolving Complex.

The generation of the diisopropyl nitroxide radical during the reaction of lithium diisopropylamide with α-fluoroacetate esters

The formation of an EPR active species was observed when lithium diisopropylamide (LDA) was employed as the base for deprotonation of fluoroacetates. The EPR of a glass prepared from the reaction mixture showed an intense signal. The solution EPR spectrum of the sample was independent of temperature, and exhibited a hyperfine structure which allowed identification of the radical species as diisopropyl nitroxide. Generation of the enolate by metalation of ethyl fluoroidoacetate gave no evidence for autoxidation occurring in the absence of diisopropylamine.

The EPR detection of foods preserved with the use of ionizing radiation

Solid constituents extracted from irradiated foods have been examined by the epr (esr) spectroscopy. It has been proved that some epr active species produced by radiation in foods are specific and stable enough to be used for the detection of irradiation treatment. The most promising results have been obtained with bones extracted from frozen raw meat (beef, pork, poultry and fish), with seeds of fruits (dates and figs), with dried mushrooms, gelatin and macaroni.

Rapid kinetics of the EPR-active species formed during initial iron uptake in horse spleen apoferritin.

The molecular mechanism of oxidative deposition of iron in ferritin is incompletely understood. In this study, EPR-active species produced during ferritin reconstitution (10-50 Fe/protein) from the apoprotein, Fe2+, and O2 have been investigated using rapid-mixing freeze-quench techniques and EPR spectroscopy. Species studied include a monomeric Fe(3+)-protein complex (g' = 4.3), a mixed-valent Fe(2+)-Fe3+ complex (g' = 1.87), and a newly observed radical with axial symmetry (g parallel = 2.042, g perpendicular = 2.0033), all apparent intermediates formed during the first second of iron oxidation. The monomeric Fe(3+)-protein complex is the principal EPR-observable product of iron(II) oxidation and is produced quantitatively in the first phase of the reaction with the mixed-valent species and the radical formed at slower rates. The initial rate of formation of the monomeric complex (and the radical) is first-order in Fe2+ concentration, consistent with a mechanism in which iron oxidation occurs in a one-electron step(s) with H2O2 being the final product of O2 reduction. A 1:1 relationship between the disappearance of the monomeric Fe(3+)-protein complex and the formation of the mixed-valent Fe(2+)-Fe3+ species was observed in the early phase of the reaction, indicating that the latter is derived from the former and not from the one-electron oxidation of a preformed Fe(2+)-Fe2+ dimer. The g-factors and rapid EPR relaxation properties of the transient radical suggest that it is associated with an Fe2+ (or Fe3+) center but its identity and possible functional role in iron oxidation are unknown.

Transient hole traps in PZT

Abstract Pb(Zr1-xTix)O3 (PZT) that has been equilibrated with an oxidizing atmosphere contains a stoichiometric excess of oxygen, and, as a result, is a p-type semiconductor at high temperatures. It becomes an insulator near room temperature because of the strong trapping of holes by cation vacancies and/or acceptor impurity centers. The trapped holes are not detected by electron paramagnetic resonance suggesting that they are trapped in diamagnetic pairs according to the so-called “negative U” effect. Illumination by band gap light produces two metastable EPR active species, identified as Pb+3 and Ti+3. Near room temperature these centers gradually decay due to recombination and the drift of holes to deep, stable centers. The transient trapping of holes at shallow centers delays the return to the stable, insulating state, and also leads to a thermally-activated hole mobility. The trapping of holes as Pb+3 centers is attributed to the electronic structure of the Pb+2 ion with its “inert pair” 6s2 electrons.

Induction of iron-derived EPR signals in murine cancers by nitric oxide. Evidence for multiple intracellular targets.

The cell-mediated immune response to syngeneic tumors activates the cytokine-inducible nitric oxide synthase. We observed that syngeneic murine tumors exhibited EPR signals related to iron-nitrosyl complex formation. Three different EPR active iron-nitrosyl species were observed, an Fe(RS)2(NO)2 signal and two differentiable heme-nitrosyl complexes. Hemoglobin assays showed that the heme-nitrosyl signals were not derived from contaminating hemoglobin. Signal amplitudes were attenuated in mice treated with N omega-mono-methyl-L-arginine (MLA), an inhibitor of nitric oxide synthase. Tumors grown in vivo contained EPR signals while those grown in culture without continuing cytokine stimulation lost the signals after a few days. Cultured cells that were treated with cytokines, or that were cocultivated with cytokine-activated macrophages, regained EPR active complexes. These results show that the cell-mediated immune response to syngeneic tumors involves the induction of nitric oxide synthase. While nitric oxide synthesis is induced in both tumor infiltrating macrophages and in the tumor cells themselves, only tumor cells contributed to formation of heme-nitrosyl complexes. This result indicates the presence of a novel intracellular target for NO within tumor cells.

DRS UV-Vis and EPR spectroscopy of hydroperoxo and superoxo complexes in titanium silicalite

The most important spectroscopic features in the UV-Vis of framework and extraframework Ti(IV) in titanium silicalite are briefly summarized. The spectroscopic manifestations of the complexes formed by framework Ti(IV) in presence of H2O2 are reported. The formation of EPR active species is also considered.

Spectroscopic Studies of Isopenicillin N Synthase: A Mononuclear Nonheme Fe2+ Oxidase with Metal Coordination Sites for Small Molecules and Substrate

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the β-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mossbauer spectra of the 57Fe-enriched enzyme exhibit parameters (δ = 1.30 mm/s, Δ EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter δ (δ = 1.10 mm/s, ΔEQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme·ACV·NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme· ACV·NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme·ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.

Magnetic properties and EPR spectroscopy of molecular metal clusters

The magnetic properties of molecular metal cluster compounds resemble those of small metal particles in the metametallic size regime. Even-electron metal carbonyl clusters with 10 or more metal atoms are paramagnetic, because their frontier orbital separations of less than 1 eV lead to high-spin electronic configurations. The rhodium cluster [Rh17S2(CO)32]3− gives EPR below 200 K with g = 2.04, the first example of this type of paramagnetism in an even-electron carbonyl cluster of this 4d metal. Its spectral parameters are compared with those of osmium carbonyl clusters and some significant differences highlighted. Attempts have also been made to generate radical cations from lowernuclearity, diamagnetic molecular clusters such as Rh6(CO)16 by chemical oxidation in sulphuric acid. An EPR active species (g = 2.09) believed to be [Rh6(CO)16]+ has been obtained.

EPR-detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800).

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.