Abstract
Some properties of a nickel species (NiC) in Desulfovibrio gigas hydrogenase (ferredoxin:H + oxidoreductase, EC 1.18.99.1), which is associated with the activated state, are described. At temperatures above 20 K this species yields an ESR spectrum at g = 2.19, 2.16, 2.01, but at lower temperatures the spectrum changed into a fast-relaxing species with a complex lineshape. Substitution of the enzyme with 61Ni shows that the complex spectrum is associated with nickel. The splitting was correlated with the presence of a broad ESR signal which probably originates from the reduced [4Fe-4S] clusters. It did not correlated with the reduced [3Fe- xS] cluster. These results indicate that the complex spectrum is due to splitting of the NiC spectrum by spin-spin interaction with the [4Fe-4S] cluster or clusters. The NiC species was light-sensitive in frozen samples, and underwent a change in spectrum which was reversed in the dark at temperatures above 200 K. Treatment of the enzyme with carbon monoxide in the presence of hydrogen induced a new type of ESR signal, which disappeared on removal of either hydrogen or carbon monoxide. The NiC species represents an intermediate oxidation state of the enzyme. The midpoint redox potentials, estimated by mediator titrations under controlled hydrogen/argon gas mixtures, were shown to be strongly pH-dependent. The values at pH 7.0 were estimated to be −270 mV (−120 mV/pH unit) for the appearance of the NiC ESR signal and −390 mV (−60 mV/pH unit) for its disappearance. The midpoint potential of the broad ESR signal was estimated to be −350 (−60 mV/pH unit). Possible schemes for redox states of the various nickel species are discussed.
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