D.V. DerVartanian

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibriogigas

Abstract A new and improved method for the purification of the periplasmic hydrogenase from Desulfovibrio gigas is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ∼2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.

Unambiguous identification of the nickel EPR signal in 61Ni-enriched Desulfovibrio gigas hydrogenase

Summary A highly active hydrogenase from Desulfovibrio gigas (sp. act. 440 μmoles H 2 evolved/min. mg) was purified from cells grown in 61 Ni enriched medium. The nuclear spin (I = 3/2) of 61 Ni induces hyperfine structure in the EPR spectra of purified hydrogenase, unequivocally identifying the previously observed signal as a Ni(III) species (LeGall, J., Ljungdahl, P., Moura, I., Peck, H.D. Jr., Xavier, A.V., Moura, J.J.G., Teixeira, M., Huynh, B.H. and DerVartanian, D.V., (1982) Biochem. Biophys. Res. Commun. 106 , 610–616). Samples reduced under hydrogen also show hyperfine structure suggesting the presence of a transient Ni(III) species in the reduced active state of the enzyme.

13C and 61Ni isotope substitutions confirm the presence of a nickel(III)-carbon species in acetogenic CO dehydrogenases

The nickel-containing CO dehydrogenases from Acetobacterium woodii and Clostridium thermoaceticum were studied by EPR spectroscopy in order to define the components involved in the EPR spectrum obtained by reaction of the enzymes with the substrate, CO. Using isotopic substitution techniques, these experiments unequivocally establish that a nickel-carbon species is involved in the g = 2.08, 2.02 EPR signal. Comparing the 61Ni- and 59Ni-substituted enzymes, the g = 2.08 component of the resonance was found to be mainly due to nickel with a smaller contribution by the carbon species. Reaction of the CO dehydrogenase with [13C]CO versus [12C]CO showed that a carbon species, formed from CO, was the major contributor to the g = 2.02 EPR signal. In addition, the oxidized CO dehydrogenase was found to exhibit a Ni (III) EPR signal analogous to that of the hydrogenases from the methanogenic and sulfate-reducing bacteria.

Electron paramagnetic resonance and light absorption studies on c-type cytochromes of the anaerobic sulfate reducer desulfovibrio

Abstract 1. 1. EPR and optical studies provide for differentiation of the c-type cytochromes examined into three groups with different heme contents. The three groups consist of: the mono-heme type, cytochrome c-553; cytochromes c3 and c′3 containing at least two hemes per molecule; and cytochromes cc3 and cc′3 which contain at least four hemes per molecule. This division into three groups agrees with a previous similar grouping based on amino acid composition and/or sequence. 2. 2. EPR studies suggest that heme-heme interaction is manifest in the ferric state of cytochrome c3. At about a half-reduced state, an EPR-detectable intermediate with a decreased degree of interaction between hemes is observed. 3. 3. Based on EPR and light absorption changes, cytochrome c3 is extremely stable in 8 M urea in the ferric state. However, repeated reduction and reoxidation in the presence of 8 M urea results in the apparent conversion of the multi-heme system to a mono-heme system. 4. 4. These and other observations suggest a conformation change involving a possible reorientation of the multi-heme moieties in the partially or fully-reduced tate. Depending on the reactant(s) present this process may be reversible.

EPR evidence for nickel-substrate interaction in carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Abstract Carbon monoxide dehydrogenase from Clostridium thermoaceticum contains two different Fe 4 S 4 rhombic-type EPR resonances with g-values at 2.04, 1.94, 1.90 and 2.01, 1.86, 1.75, respectively. The enzyme after reacting with CO or HCO 3 − /CO 2 also reveals in EPR signal at g = 2.07 and 2.02. This signal, readily observed at 95K, is attributed to a Ni(III) interaction with a radical species formed from CO or HCO 3 − /CO 2 .

Evidence for the involvement of non-heme iron in the active site of hydrogenase from Desulfovibrio vulgaris.

Abstract 1. 1. Hydrogenase (hydrogen: cytochrome c 3 oxidoreductase) of high specific activity has been purified to near homogeneity as judged by disc electrophoresis and in the analytical ultracentrifuge, and found to contain 3.5 iron and 3.2 labile-sulfide atoms per molecule (of mol. wt. 60 000). 2. 2. Hydrogenase was found to exist in a dimer form of mol. wt. 60 000 and a monomer form of 30 000. The dimer—monomer forms were concentration-dependent with the dimer form favored at high protein concentrations. 3. 3. Light absorption and EPR studies definitely indicate a redox functional iron-sulfide unit at the active site. On reduction with H 2 , a g = 1.86 EPR absorption was observed. This is the Beinert “ g = 1.9” type of EPR absorption observed in other iron-sulfide proteins such as plant and bacterial ferredoxins.

The bacterial nitrate reductases: EPR studies on nitrate reductase A from Micrococcus denitrificans

EPR studies on nitrate reductase indicate at 80 °K a signal at g=1.985 and g=2.045 attributable to Mo(V). At 15 °K a signal is observed at g=2.016 due to Fe(III) in an unknown symmetry. The reaction of nitrate reductase with nitrate, nitrite or azide results in changes of the Mo(V) signal. Quantitation of the Mo(V) signal intensity accounts for 15% of the chemical molybdenum content. A similar quantitation of the Fe(III) signal intensity indicates equimolarity with enzymatically reducible g=1.9-type resonance. Enzymatic reduction of nitrate reductase and nitrate with sodium dithionite plus benzyl viologen or molecular hydrogen plus hydrogenase results in the disappearance of the initial Mo(V) and Fe(III) signals and the appearance of a temperature-sensitive iron-sulfide absorption with g-values at 2.057, 1.947 and 1.881 when measured at 15 °K. Chemical reduction of nitrate reductase with sodium dithionite also results in loss of the initial Mo(V) and Fe(III) signals but gives a more complex iron-sulfide signal consisting of two different types of iron-sulfide systems. Quantitation of both types of iron-sulfide system indicated they are present in approx. equal concentrations. These two iron-sulfide systems may differ in redox potential. These studies support the involvement of iron and molybdenum in nitrate reductase.

EPR determination of the oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio vulgaris.

EPR spectroscopy in conjunction with oxidation-reduction potentiometry has been used to determine the half-reduction potentials of the four hemes of cytochrome c3. As predicted, the four hemes of cytochrome c3 have different mid-point potentials. The Em values are: Heme I,--284 mV; Heme II,--310 mV; Heme III,--324 mV and Heme IV,--319 mV. The n-values in each case was near one.

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions

Abstract Nitrite reductase (cytochrome cd ) from T. denitrificans has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from P. aeruginosa . EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both c - and d -hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a 14N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant. To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.

Electron paramagnetic resonance studies on the nature of hemoproteins in nitrite and nitric oxide reduction.

Abstract Nitrite and nitric oxide reductases and c -type cytochromes were isolated from nitrate-grown Pseudomonas perfectomarinus . EPR measurements indicate the direct participation of c -type cytochromes in the reduction of nitrite to nitric oxide. The EPR resonance of heme-NO complexes is observed with g -values at 2.03 and 2.10 with 14 N hyperfine splitting of 16.2 Gauss. Quantitative EPR measurements indicate that all the c -type cytochromes involved in nitrite reduction are in the heme-NO complex forms and that these complexes account for about 15% of the total nitric oxide observed. A different c -type cytochrome was found to be involved in the reduction of NO to N 2 O with the major g -value of the heme-NO complex at 2.06. A time-dependent broadening of EPR was noted.