Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum. Kinetic investigations of cross-reactions as a probe of the enzyme mechanism.
In combination with the Mo-Fe protein of nitrogenase from Klebsiella pneumoniae, the Fe protein of nitrogenase from Clostridium pasteurianum forms an active enzyme with novel properties different from those of either of the homologous nitrogenases. The steady-state rates of reduction of acetylene and H+ are 12% of those of the homologous system from C.pasteurianim. Acetylene reductase activity exhibited an approx. 10min lag at 30 degrees C before the rate of reduction became linear, consistent with a once-only activation step being necessary for acetylene reduction to occur. No such lag was observed for H2 evolution. The activity with N2 as a reducible substrate was very low, implying that acetylene reductase activity is not necessarily an accurate indication of nitrogen-fixing ability. This is of particular relevance to studies on mutant and agronomically important organisms. Stopped-flow spectrophotometric studies showed unimolecular electron transfer from the Fe protein to the Mo-Fe protein to occur at the same rate (k2 = 2.5 X 10(2)s-1) and with the same dependence on ATP concentration (apparent KD = 400 muM) as with the homologous Klebsiella nitrogenase. However, an ATP/2e ratio of 50 was obtained for H2 evolution, indicating that ATP hydrolysis had been uncoupled from electron transfer to substrate. These data indicate that ATP has at least two roles in the mechanism of nitrogenase action. The combination of the Mo-Fe protein of nitrogenase of C.pasteurianim and the Fe protein of K.pneumoniae were inactive in all the above reactions, except for a weak adenosine triphosphatase activity, 0.5% of that of the homologous K.pneumoniae system.
D000114:Acetylene; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D003013:Clostridium / Q000201:enzymology*; D005288:Ferredoxins / Q000032:analysis; D006859:Hydrogen; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008274:Magnesium; D008982:Molybdenum; D009591:Nitrogenase / Q000378:metabolism*
D005288:Ferredoxins; D006859:Hydrogen; D008982:Molybdenum; D000255:Adenosine Triphosphate; D009591:Nitrogenase; D000251:Adenosine Triphosphatases; D008274:Magnesium; D000114:Acetylene