Catalysis of exchange of terminal phosphate groups of ATP and ADP by purified nitrogenase proteins.
A crude Azotobacter nitrogenase complex contained a highly active adenylate kinase which caused rapid equilibration of AMP, ADP, and ATP. Purified molybdenum-iron protein preparations also contained measurable adenylate kinase activity which could be removed by adsorption and elution from hydroxylapatite. Independent of adenylate kinase, the highly purified molybdenum-iron protein from both Klebsiella pneumoniae and Azotobacter chroococcum catalyzed the exchange of [32P]orthophosphate with the terminal phosphate of ATP or ADP. The exchange labeling of ATP was stimulated by ferricyanide ion due to the inhibition of ATP hydrolysis linked to substrate reductions which cannot occur in the absence of reductant. This exchange reaction is responsible for reported ATP synthesis by crude nitrogenase preparations. Binding of ATP labeled with 32P in the terminal phosphate group was measured directly with concentrated solutions of the molybdenum-iron nitrogenase protein from K. pneumoniae by the column gel filtration method. The protein was saturated with ATP at a calculated ratio of 4.0 +/- 0.3 mol ATP/mol protein; half-saturation of 220 microM protein occurred at 600 +/- 100 microM. The interactions between adenine nucleotides and the molybdenum-iron protein suggest the involvement of the nucleotides in a role distinct from the established reactions with the iron protein of the nitrogenase complex. A dual role for ATP in the reduction of dinitrogen by isolated nitrogenase proteins would be consistent with the existence of binding sites for ATP on both the iron and molybdenum-iron proteins.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism; D001395:Azotobacter / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D005288:Ferredoxins / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008274:Magnesium; D008983:Molybdoferredoxin / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*
D005288:Ferredoxins; D008983:Molybdoferredoxin; D010710:Phosphates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009591:Nitrogenase; D000263:Adenylate Kinase; D008274:Magnesium