Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre.
We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per alpha2beta2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.
Yousafzai
F K
FK
Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, U.K.
Eady
R R
RR
D004789:Enzyme Activation; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008983:Molybdoferredoxin / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D011487:Protein Conformation
D008983:Molybdoferredoxin; D009591:Nitrogenase