Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study.

The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium Vinosum can exist in three different redox states. In the most oxidized state (Nia-S) the nickel is divalent. The most reduced state (Nia-SR) likewise has Ni2+, while the intermediate state (Nia-C*) has Ni3+. The transitions between these states have been studied by stopped-flow Fourier transform infrared spectroscopy. It is inferred from the data that the Nia-S f Nia-C* and Nia-C* f Nia-SR transitions induced by dihydrogen require one of the [4Fe-4S] clusters to be oxidized. Enzyme in the Nia-S state with all of the iron-sulfur clusters reduced reacts with dihydrogen to form the Nia-SR state in milliseconds. By contrast, when one of the cubane clusters is oxidized, the Nia-S state reacts with dihydrogen to form the Nia-C* state with all of the iron-sulfur clusters reduced. The competition between dihydrogen and carbon monoxide for binding to the active site was dependent on the redox state of the nickel ion. Formation of the Nia-SâCO state (Ni2+) by reacting CO with enzyme in the Nia-SR and Nia-S states (Ni2+) is considerably faster than its formation from enzyme in the Nia-C* (Ni3+) state. Excess oxygen converted hydrogen-reduced enzyme to the inactive Nir* state within 158 ms, suggesting a direct reaction at the Ni-Fe site. With lower O2 concentrations the formation of intermediate states was observed. The results are discussed in the light of the present knowledge of the structure and mechanism of action of the A. Vinosum enzyme