Nitrogenase IX. Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro

Abstract

Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays. Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase. The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A. and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity. In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase. The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities. Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed.