Kinetics and mechanism of the nitrogenase reaction

The basic mechanism of nitrogenase with the use of dithionate as an electron donor for the iron protein involves the following steps (Thomeley and Lowe, 1985 Likhtenshtein, 1988a Burgess and Lowe, 1996 Smith, 1999 Seefeldt and Dean, 1997 Rees and Howard, 2000 Syrtsova and Timofeeva, 2001) 1) reduction of Fe-protein with flavodoxin or dithionate and attachment of two ATP molecules to the protein, 2) formation of a complex between the reduced FeP with two bound ATP molecules and FeMo-protein, 3) electron transfer between the reduced [Fe4S4] cluster of FeP to the P-cluster ofFeMoP coupled to the ATP hydrolysis, 4) electron transfer from P-cluster to 

FeMoco, 5) dissociation of the FeP-FeMoP complex accompanied by re-reduction of FeP and exchange of ATP for ADP, and repetition of this circle until a sufficient number electrons and protons have been accumulated in FeMoco so that the available substrate can be reduced. 

The results of studying the kinetics of electron transfer from the Fe protein to the Mo—Fe-protein with the artificial electron donor, dithionite (Thomeley and Lowe, 1985), are presented in Fig.3.5. 

According to recent data, the property of dithionite as an electron donor for nitrogenase is different from that of the natural donor flavodoxin (Burgess and Lowe, 1996). Flavodoxin from Azotobacter vinelandii has the redox potential equal to -0.515 V for the reversible transition between the semiquinone and hydroquinone forms of flavodoxin. Unlike dithionite, flavodoxin can reversibly reduce the [Fe4S4]+l cluster Av2 by one electron to the [Fe4S4]° state in which all iron ions exist in the ferrous form. It is assumed that, under natural conditions, two electrons can transfer from Av2 to Avl. Flavodoxin reduces both Av2 bound to Avl and free Av2 in a solution. The apparent rate constants of these reactions are 400 s 1 and 1000 s 1, respectively (Duyvis et al. 1998). 

It has been established (Druzhinin et al., 1995) that, in the case of the photochemical eosin—NADH system and dithionite concentrations not higher than 4 10 4M, the Avl Av2 complex (1 1) is enzymatically active. The slow process (0.1 s 1 k 2.0 102 s 1 at 20 °C) of nitrogenase dissociation to the Avl and Av2 components was not observed. The rate constant of a second order for the reduction of Av2 in the nitrogenase complex with the eosin—NADH photodonor was found to be equal to 1.1 107 M 1 s 1. 

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