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Kinetic Studies of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase

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journal contribution
posted on 12.08.2014, 00:00 by Anurag P. Srivastava, David B. Knaff, Pierre Sétif
A flash photolysis study of electron transfer (ET) kinetics from reduced ferredoxin (photoreduced by Photosystem I) to the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been carried out. In the presence of nitrate, under conditions where only a single electron is transferred to nitrate reductase, the rate of enzyme reduction shows a biphasic concentration dependence: At low enzyme concentrations the dependence is approximately linear, with an estimated second-order rate constant of 7.4 ± 0.8 × 107 M–1 s–1; at concentrations above 2 μM, the rate increases nonlinearly to an asymptotic value of approximately 300 s–1, indicating the presence of a rate-limiting step in the process. The spectrum of the one-electron reduced enzyme suggests that Mo centers are largely reduced with a minor contribution of iron–sulfur cluster reduction. Under conditions favoring two-electron reduction of the enzyme, the redox difference spectrum can be accounted for by the oxidation of two reduced ferredoxins, suggesting that the enzyme has completed one full catalytic cycle. The spectral changes observed in the absence of nitrate are significantly different from those seen in the presence of nitrate. Experiments in the absence of nitrate revealed that the singly reduced enzyme exhibits different absorption characteristics and reoxidation kinetics, compared to those observed with nitrate present, and exhibits a much faster binding by reduced ferredoxin than the oxidized enzyme. The implications of these observations for understanding the enzyme mechanism are discussed.

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