title: The role of MgATP hydrolysis in nitrogenase catalysis.
abstract: Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye-oxidized nitrogenase proteins it can be excluded that reductant-independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant-dependent and reductant-independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s-1 at pH 7.4 and 22 degrees C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant-independent ATPase activity is only about 6% of that of reductant-dependent ATPase activity. While the reductant-dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant-independent ATPase activity yields hyperbolic saturation curves. To account for these results it is proposed that the rate-limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Cordewener
authors.forename: M
authors.initials: M
authors.lastname: Krüse-Wolters
authors.forename: H
authors.initials: H
authors.lastname: Wassink
authors.forename: H
authors.initials: H
authors.lastname: Haaker
authors.forename: C
authors.initials: C
authors.lastname: Veeger
mesh_terms: D001395:Azotobacter / Q000201:enzymology; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002384:Catalysis; D004579:Electron Transport; D005296:Ferrous Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008667:Metalloproteins / Q000378:metabolism; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013696:Temperature
chemical_list: D005296:Ferrous Compounds; D008667:Metalloproteins; D009591:Nitrogenase; D017301:Ca(2+) Mg(2+)-ATPase