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Abstract
The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium 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 (approximately 4,000 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 (1,400-2,000 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). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.
Highlights
A direct role for nucleotides in nitrogenase substrate reduction reactions was first demonstrated in 1964 [7] when it was discovered that the addition of MgATP stimulated nitrogen fixa
Nucleotide Hydrolysis by Nitrogenase—Hydrolysis of MgATP by the Fe protein-MoFe protein complex can occur in the absence of electron transfer or coupled to electron transfer
The Km values measured for the hydrolysis of each nucleotide examined were near the value for ATP
Summary
(Received for publication, November 2, 1999, and in revised form, December 3, 1999). Matthew J. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF4Ϫ resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF4Ϫ1⁄7MgNDP The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed. Evidence is presented that nitrogenase catalyzes the hydrolysis of purine and pyrimidine nucleotides (MgGTP, MgITP, and MgUTP) other than ATP at significant rates. These rates were sensitive to the oxidation state of the nitrogenase proteins, with an oxidized enzyme having much greater rates of hydrolysis than the reduced enzyme. The nature of the interactions of these other nucleotides with nitrogenase are examined, and the implications of these findings in understanding roles for nucleotides in the nitrogenase mechanism are discussed
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