Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas.

J J Moura,J LeGall,H D Peck,A V Xavier,B H Huynh,I Moura,M Teixeira,D V DerVartanian

doi:10.1016/s0021-9258(17)39440-1

J J Moura, J LeGall

Open Access

https://doi.org/10.1016/s0021-9258(17)39440-1

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Abstract

Desulfovibrio gigas hydrogenase (EC 1.12.2.1) is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.

Highlights

Electron Paramagnetic ResonanceStudies on the Mechanism of Activation and the Catalytic Cycle ofthe Nickel-containing Hydrogenase fromDesulfovibriogigas*
This assignment was confirmed by the observation to the intermediate redospxecies generated during the of hyperfine coupling in 61Niisotopic labeled hydrogenase and catalyticcycle of the enzyme antodthe midpoint redox by comparison with model nickel compounds (6,lO).A minor potentials associated
A comprehension of the mechanism of action of hydrogenase can only be achieved by a full characterization of the structure andphysicochemicalproperties of the redox centers as well as their interaction andbehavior during the catalytic cycle

Summary

MATERIALS ANDMETHODS

Growth of the Organisms and Purification of Hydrogenase and Cytochrome c3-D. gigas was grown on a medium as described by Le q> -. After a long incubation (36-48 h) under hydrogen, an EPR silent state isobtained when measured at 77 K (Fig. 3 E ) .At low temperature (below 15 K), EPR signals typical of [Fe4S411+clusters are observed This sequence of events can be reproducibly reversed by anaerobically oxidizing the reduced sample under argon and completely repeated by exposing the reoxidized sample to hydrogen. Other experimental conditions: modulation amplitude, 1 millitesla; microwave power, milliwatts; microwave frequency, 9.41 GHz. oxidation products; as we have already pointed out, a radical species is observed during the reductive pattern of chrome c3-The effects of cytochrome c3 on the redox pattern the enzyme (Figs. and 8). EPR spectra representing the time course of redox cycling reduced with excess amounts of dithionite, the Ni-signal C of native hydrogenase in the presence of ferricytochrome c3 and the g = 2.21 signal disappear, and, at low temperature, were recorded at 77 K.

CONCLUSIONS

Nickel Chemistry in the Context of Its Biological Role

Hypothesis B

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