Nitrogenase of Azotobacter vinelandii: Kinetic analysis of the Fe protein redox cycle

Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) w hich are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by Mg ATP hydrolysis and is repeated until the substrates are completely reduced. The rate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obl igatory for the reduction of the Fe protein [Thorneley, R. N. F., and Lowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experi ments with dithionite as the reductant. We also tested besides dithionite f lavodoxin hydroquinone, a physiological reductant. Two models could describ e the experimental data of the reduction by dithionite. The first model, wi th no reduction of Fe protein bound to MoFe protein, predicts a rate of dis sociation of the protein complex of 8.1 s(-1). This rate is too high to be the rate-limiting step of the Fe protein cycle (k(obs) = 3.0 s(-1)). The se cond model, with reduction of the Fe protein in the nitrogenase complex, pr edicts a rate of dissociation of the protein complex of 2.3 s(-1), which in combination with reduction of the nitrogenase complex can account for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinon e (155 mu M) was the reductant, the rate of reduction of oxidized Fe protei n in the nitrogenase complex (k(obs) approximate to 400 s(-1)) was 100 time s faster than the turnover rate of the cycle with flavodoxin as the reducta nt (4 s(-1)). Presteady-state electron uptake experiments from flavodoxin h ydroquinone indicate that before and after reduction of the nitrogenase com plex relative slow reactions take place, which limits the rate of the Fe pr otein cycle. These results are discussed in the context of the kinetic mode ls of the Fe protein cycle of nitrogenase.