Evidence for multiple steps in the pre-steady-state electron transfer reaction of nitrogenase from Azotobacter vinelandii

Duyvis, M.G.; Mensink, Richard E.; Wassink, Hans; Haaker, H.

doi:10.1016/s0005-2728(97)00006-6

Cited by 12 publications

(4 citation statements)

References 28 publications

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“…Samples were injected in pre‐heated Ar, flushed EPR tubes and subsequently rapidly frozen in liquid N 2 /isopentane. The freezing time was 0.5 s [12].…”

Section: Methodsmentioning

confidence: 99%

Hyperthermophilic redox chemistry: a re‐evaluation

et al. 1998

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The redox chemistry of Pyrococcus furiosus rubredoxin and ferredoxin has been studied as a function of temperature in direct voltammetry and in EPR monitored bulk titrations. The E m s of both proteins, measured with direct voltammetry, have a normal (linear) temperature dependence and show no pH dependence. EPR monitoring is not a reliable method to determine the temperature dependence of the E m : upon rapid freezing the proteins take their conformation corresponding to the freezing point of the solution.z 1998 Federation of European Biochemical Societies.

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“…Samples were injected in pre‐heated Ar, flushed EPR tubes and subsequently rapidly frozen in liquid N 2 /isopentane. The freezing time was 0.5 s [12].…”

Section: Methodsmentioning

confidence: 99%

Hyperthermophilic redox chemistry: a re‐evaluation

et al. 1998

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“…FeMoco is generally considered to be the substrate reduction site. The P-cluster might be involved in electron transfer from the Fe protein to FeMoco (2)(3)(4). Structural models for FeMoco and the P-cluster have been proposed, on the basis of crystallographic analysis of the MoFe protein (5)(6)(7).…”

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confidence: 99%

Nitrogenase of Azotobacter vinelandii: Kinetic Analysis of the Fe Protein Redox Cycle

1998

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Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by MgATP 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 obligatory 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 experiments with dithionite as 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, with no reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation 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 (kobs = 3.0 s-1). The second model, with reduction of the Fe protein in the nitrogenase complex, predicts 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 hydroquinone (155 microM) was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs approximately 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 after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle of nitrogenase.

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“…The Fe‐protein functions as an electron donor to the MoFe‐protein. This electron transfer is thermodynamically unfavorable and requires the hydrolysis of 2MgATP per electron transferred [9–13]. Apart from the critical role that the Fe‐protein plays in electron transfer to the MoFe‐protein in a MgATP‐dependent reaction, the Fe‐protein also has at least two additional functions.…”

Section: Introductionmentioning

confidence: 99%