Nitrogenase of Azotobacter vinelandii: Kinetic Analysis of the Fe Protein Redox Cycle
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- 作者：Martina G. Duyvis ; Hans Wassink ; and Huub Haaker
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：December 15, 1998
- 年：1998
- 卷：37
- 期：50
- 页码：17345 - 17354
- 全文大小：157K
- 年卷期：v.37,no.50(December 15, 1998)
- ISSN：1520-4995
文摘
Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumedto associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe proteincycle, is driven by MgATP hydrolysis and is repeated until the substrates are completely reduced. Therate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obligatory for the reduction of the Fe protein [Thorneley, R. N. F., andLowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experiments with dithioniteas the reductant. We also tested besides dithionite flavodoxin hydroquinone, a physiological reductant.Two models could describe the experimental data of the reduction by dithionite. The first model, withno reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation of the protein complexof 8.1 s-1. This rate is too high to be the rate-limiting step of the Fe protein cycle (kobs = 3.0 s-1). Thesecond model, with reduction of the Fe protein in the nitrogenase complex, predicts a rate of dissociationof the protein complex of 2.3 s-1, which in combination with reduction of the nitrogenase complex canaccount for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinone (155 
M)was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs 
400 s-1)was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and afterreduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Feprotein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle ofnitrogenase.
M)was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs 400 s-1)was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and afterreduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Feprotein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle ofnitrogenase.