Klebsiella pneumoniae nitrogenase: Formation and stability of putative beryllium fluoride-ADP transition state complexes

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component pr oteins of Klebsiella pneumoniae nitrogenase, with BeF3- and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1 .{Kp2 .(MgADP . BeFx)(2)}(2). At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 comp lex, the partially active 1:1 Kp1 . Kp2 (MgADP . BeFx)(2) complex, and acti ve nitrogenase components was demonstrated. The inhibition was reversible s ince incubation of the 1:1 complex in the absence of MgADP and beryllium re sulted in complete restoration of activity over 30 h. Under pseudo-first-or der conditions with regard to nitrogenase components and MgADP, the kinetic s of the rate of inhibition with increasing concentrations of BeF3- showed a square dependence on [BeF3-], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1 Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 com plex from free Kp1. Deconvolution of the equilibrium profiles gave concentr ations of the components allowing constants for their formation of 2.1 x 10 (6) and 5.6 x 10(5) M-1 to be calculated for the 1:1 and 2:1 complexes, res pectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites u noccupied. The value for K-1 we obtain from this study is comparable with t he value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mi xture containing 20 mu M nitrogenase components showed 97% of the Kp2 prese nt initially to be complexed. These data provide the first unequivocal demo nstration that Fe protein preparations which may contain up to 50% of a spe cies of Fe protein defective in electron transfer is nevertheless fully com petent in complex formation with MoFe protein.