The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas

A new and improved method for the purification of the periplasmic hydrogenase from Desulfovibrio gigas is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni (III) signal with g-values at 2.31, 2.20 and similar to 2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni (III) signals reappeared along with additional signals attributed to multiple Ni (III) species. It is proposed that Ni is an important functional unit in this hydrogenase.