A pulsed EPR study of redox-dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase.

The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni-A) and H2-reduced active (Ni-C) states, both in H2O and 2H2O solutions. Fourier transforms of the 3-pulse ESEEM, taken at 8.7 GHz, for Ni-A and Ni-C in H2O contained similar peaks with narrow linewidths at frequencies of 0.4, 1.2 and 1.6 MHz, and a broader peak centered at 4.5 MHz. At 11.6 GHz, the low frequency components showed small field-dependent shifts, while the high frequency component was shifted to 5.1 MHz. These results are consistent with the presence of 14N, possibly from imidazole, coupled to the nickel centre. In 2H2O, Ni-A was shown to be inaccessible for exchange with solvent deuterons. In contrast, Ni-C was accessible to solvent exchange, with a deuterium population being in close proximity to the metal ion. Thus, the nickel environment of the active protein is different from that in the oxidized or unready state. On illumination of Ni-C, although EPR changes are seen, 14N coupling remains, and for the 2H2O sample, deuterium coupling is also retained.