title: Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.
abstract: The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.
authors.affiliation: AFRC Unit of Nitrogen Fixation, University of Sussex, Falmer, Brighton, U.K.
authors.forename: G A
authors.initials: GA
authors.lastname: Ashby
authors.forename: R N
authors.initials: RN
authors.lastname: Thorneley
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D004227:Dithionite / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D013053:Spectrophotometry
chemical_list: D004227:Dithionite; D000255:Adenosine Triphosphate; D009591:Nitrogenase