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

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 H-2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Ni-a-SR state) to the Ni-a-C* state was completed in less than 8 ms, a rate consistent with the H-2-evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Ni-a-SR state did not react with CO. It was discovered, contrary to expectations, that the Ni-a-C* state did not react with CO when reactions were performed in the dark. When H-2 was replaced by CO, a Ni-a-C* EPR signal appeared within 11 ms; this was also the case when H-2 was replaced by Ar. With CO, however, the Ni-a-C* state decayed within 40 ms, due to the generation of the Ni-a-S . CO state (the EPR-silent state of the enzyme with bound CO). The Ni-a-C* state, induced after 11 ms by replacing H-2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR-detectable state with CO bound to nickel (Ni-a*. CO) by illumination at 30 K (evoking the Ni-a-L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Ni-a-C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Ni-a-L* and the Ni-a*. CO states is formally monovalent.