Nitrogenase of Klebsiella pneumoniae nifV mutants. Investigation of the novel carbon monoxide-sensitivity of hydrogen evolution by the mutant enzyme

Abstract

The MoFe protein of nitrogenase from K. pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalyzed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalyzed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoichiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. CO did not affect the rate of phosphate release from ATP. The ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH 6.3 to 82% at pH 8.6. Inhibition at pH 7.4 (maximum 73%) was half-maximal at 0.031 matm CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction. A single high-affinity CO-binding site in the NifV- Kp1 protein may cause partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.