Purification and properties of the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

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Abstract
A soluble hydrogenase was isolated from Desulfovibrio desulfuricans (strain Norway 4) grown on Postgate''s medium. The enzyme differs significantly from a membrane-bound hydrogenase previously purified from the same organism grown on Starkey''s medium. The enzyme consisted of 2 subunits of 56 kDa (kilodalton) and 29 kDa compared with masses of 69 kDa and 27 kDa for the membrane-bound enzyme. Analysis of preparations of the soluble enzyme by various methods gave values of 5-10 Fe atoms, 6 labile S atoms and 0.45-0.8 Ni atoms/molecule. The enzyme was unusual in that it contained Se, in quantities equivalent to Ni. The highly purified active enzyme produced no ESR signals in the oxidized state. ESR signals due to a [3Fe-xS] cluster and Ni were observed only in some of the less active fractions of the enzyme, demonstrating that neither of these ESR-detectable components is a prerequisite for hydrogenase activity. Treatment of D. desulfuricans (Norway) cells with EDTA released a minor fraction with hydrogenase activity, which might indicate the presence of a periplasmic enzyme.