Activation of the nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: kinetic characterization and reductant requirement.

The requirements for and kinetics of the activation of the nickel-deficient (apo) CO dehydrogenase from Rhodospirillum rubrum by exogenous nickel have been investigated. The activation is strictly dependent upon the presence of a low-potential one-electron reductant. Sodium dithionite and reduced methylviologen (E degrees' = -440 mV) are suitable reductants, whereas reduced indigo carmine (E degrees' = -125 mV) and the two-electron reductants sodium borohydride, NADH, and dithiothreitol are ineffective in stimulating activation. The midpoint potential for activation was observed at approximately -475 mV. The ability of a reductant to stimulate activation is correlated with the reduced state of the enzyme Fe4-S4 centers. The activation follows apparent first-order kinetics in a saturable fashion, yielding a rate constant of 0.157 min-1 at saturating concentration of nickel. The initial rate at which the enzyme is activated by NiCl2 is also a saturable process, yielding a dissociation constant (KD) of 755 microM for the initial association of nickel and enzyme. Cadmium(II), zinc(II), cobalt(II), and iron(II) are competitive inhibitors of nickel activation with inhibition constants of 2.44, 4.16, 175, and 349 microM, respectively. Manganese(II), calcium(II), and magnesium(II) exhibit no inhibition of activation.