title: Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.
abstract: Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) 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 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], 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 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations 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, respectively. 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 unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.
authors.affiliation: Nitrogen Fixation Laboratory, John Innes Centre, Norwich, U.K.
authors.forename: T A
authors.initials: TA
authors.lastname: Clarke
authors.forename: F K
authors.initials: FK
authors.lastname: Yousafzai
authors.forename: R R
authors.initials: RR
authors.lastname: Eady
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001608:Beryllium / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004795:Enzyme Stability; D005459:Fluorides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D046911:Macromolecular Substances; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D013997:Time Factors
chemical_list: D046911:Macromolecular Substances; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D009591:Nitrogenase; D001608:Beryllium; D005459:Fluorides