Pre‐steady‐state kinetics of the reactions of [NiFe]‐hydrogenase from Chromatium vinosum with H2 and CO

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Abstract
Results are presented of the first rapid‐mixing/rapid‐freezing studies with a [NiFe]‐hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia‐SR state) to the Nia‐C* state was completed in less than 8 ms, a rate consistent with the H2‐evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia‐SR state did not react with CO. It was discovered, contrary to expectations, that the Nia‐C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia‐C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia‐C* state decayed within 40 ms, due to the generation of the Nia‐S·CO state (the EPR‐silent state of the enzyme with bound CO). The Nia‐C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR‐detectable state with CO bound to nickel (Nia*·CO) by illumination at 30 K (evoking the Nia‐L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia‐C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia‐L* and the Nia*·CO states is formally monovalent.

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