Nitrogenase of Klebsiella pneumoniae nifV mutants.
The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore 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 pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (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 with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.
D000114:Acetylene / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002248:Carbon Monoxide / Q000494:pharmacology; D004227:Dithionite / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008983:Molybdoferredoxin / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D008983:Molybdoferredoxin; D004227:Dithionite; D002248:Carbon Monoxide; D006859:Hydrogen; D000255:Adenosine Triphosphate; D009591:Nitrogenase; D000114:Acetylene