Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SI _r state and its light sensitivity

Abstract

The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SI _r, provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SI _a, Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SI _r was found to be light-sensitive ( T ≤ 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-SL) was shown to involve an additional transient light-induced state (Ni-SI ₁₉₆₁). Rapid scan kinetic infrared measurements provided activation energies for the transition from Ni-SL to Ni-SI _r in protonated as well as in deuterated samples. The inhibitor CO was found not to react with the active site of the Ni-SL state. The wavelength dependence of the Ni-SI _r photoconversion was examined in the range between 410 and 680 nm. Light-induced effects were associated with a nickel-centred electronic transition, possibly involving a change in the spin state of nickel (Ni ²⁺). In addition, at T ≤ 40 K the CN ⁻ stretching vibrations of Ni-SL were found to be dependent on the colour of the monochromatic light used to irradiate the species, suggesting a change in the interaction of the hydrogen-bonding network of the surrounding amino acids. A possible mechanism for the photochemical process, involving displacement of the oxygen-based ligand, is discussed.

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