Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

• Published in: The Journal of biological chemistry Vol. 275; no. 9; pp. 6214 - 6219
• Main Authors: Ryle, Matthew J., Seefeldt, Lance C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.03.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - metabolism; ADP; Aluminum Compounds - pharmacology; Azotobacter vinelandii; Azotobacter vinelandii - enzymology; Bacterial Proteins - metabolism; Clostridium - enzymology; Clostridium pasteurianum; Dithionite - metabolism; Fluorides - pharmacology; Guanosine Triphosphate - metabolism; Inosine Triphosphate - metabolism; Kinetics; magnesium GTP; magnesium ITP; magnesium UTP; molybdenum-iron protein; Molybdoferredoxin; Nitrogenase - metabolism; nucleoside diphosphates; Nucleotides - metabolism; Oxidation-Reduction; Oxidoreductases; Uridine Triphosphate - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.9.6214

The hydrolysis of ATP to ADP and Pi is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii andClostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (∼4000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1400–2000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80–230 nmol of MgNDP formed per min/mg of Fe protein). TheKm values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330–540 μm) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF4− resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF4−·MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.