Infrared spectroelectrochemical characterization of the [NiFe] hydrogenase of Desulfovibrio gigas

Electrochemically controlled titrations of the different redox states detected by IR spectroscopy of Desulfovibrio gigas hydrogenase are reported. This enzyme has in common with other metal-containing hydrogenases the existence of three intense bands; at exceptionally high frequencies that in Chromatium vinosum hydrogenase have been found to arise from two CN- and one CO (Happe, R. P.; Roseboom, W.; Pierik, A. J.; Albracht, S. P. J.; Bagley, K. A. Nature 1997, 385, 126). Here we propose a tentative assignment of these special groups to the three diatomic active site Fe ligands observed in the crystal structure of Desulfovibrio gigas hydrogenase (Volbeda, A.; Garcin, E.; Piras, C.; de Lacey, A. L.; Fernandez, V. hi.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. J. Am. Chem. Sec. 1996, 118, 12989-12996), based on their interaction with the protein environment. IR states equivalent to each one of the Ni-EPR detectable states and their corresponding one-electron reduced EPR-silent forms have been identified by in situ redox titration. The similarity between the IR titration and the reported stoichiometric reductive titrations of EPR nickel signals of D. gigas enzyme (Roberts, L. M.; Lindahl, P. A. J. Am. Chem. Sec. 1995, 117, 2565-2572) suggest that there is a strong electronic interaction between the two metal centers in the active site. The IR-spectroelectrochemical technique used here allows for further insight into the activation, inactivation, and catalytic cycles of the enzyme. The path of activation and the identity of the active states have been characterized.