Nitrogenase of Azotobacter vinelandii: kinetic analysis of the Fe protein redox cycle.

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.

Pre-steady-state kinetics of nitrogenase from Azotobacter vinelandii. Evidence for an ATP-induced conformational change of the nitrogenase complex as part of the reaction mechanism.

The pre-steady-state electron transfer reactions of nitrogenase from Azotobacter vinelandii have been studied by stopped-flow spectrophotometry. With reduced nitrogenase proteins after the initial absorbance increase at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbance decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductant-free nitrogenase proteins with MgATP leads after 20 ms to a decrease of the absorbance, which could be fitted (from 0. 05 to 1 s) with a single exponential decay with a rate constant kobs = 6.6 +/- 0.8 s-1. This reaction of nitrogenase was measured at different wavelengths. The data indicate the formation of a species with a blue shift of the absorbance of metal-sulfur clusters of nitrogenase from 430 to 360 nm. The absorbance decrease at 430 nm observed (after 50 ms) in the case of the reduced nitrogenase proteins could only be simulated well if, after the initial electron transfer from the Fe protein to the MoFe protein and before dissociation of the nitrogenase complex, an additional reaction was assumed. The rate constant of this reaction was of the same order as the rate constant of the MgATP-dependent pre-steady-state proton production by nitrogenase from A. vinelandii: kobs = 14 +/- 4 s-1 with reduced nitrogenase proteins and kobs = 6 +/- 2 s-1 with dithionite-free nitrogenase proteins (Duyvis, M. G., Wassink, H., and Haaker, H. (1994) Eur. J. Biochem. 225, 881-890). It is proposed that in the presence and absence of reductant, the observed absorbance decrease at 430 nm of nitrogenase is caused by a change of the conformation of the nitrogenase complex, as a consequence of hydrolysis of MgATP.

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase.

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50 degrees C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulfur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex with nitrogenase is isolated.

Pre-steady-state MgATP-dependent proton production and electron transfer by nitrogenase from Azotobacter vinelandii.

MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 degrees C is 14 +/- 4 s-1. No proton production with a rate constant comparable to the observed rate constant of electron transfer (kobs approximately 100 s-1) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +/- 0.3 mol H+produced/mol MoFe protein with oxidized Fe protein, and 1.1 +/- 0.1 mol H+produced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electrons(transferred)/H-uptake was calculated to be 1.2 +/- 0.2. After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (kobs approximately 35 s-1). The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein; with dithionite-reduced Fe protein a value of 3.1 +/- 0.4 mol H+produced/mol MoFe protein was found (or 0.5 +/- 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 +/- 0.05 mol H+/mol Fe protein. All reported absorbance changes were absent when the experiments were performed in a buffered solution. The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-1, which is followed by H+ production (kobs approximately 14 s-1). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases.(ABSTRACT TRUNCATED AT 400 WORDS)

The third subunit of desulfoviridin-type dissimilatory sulfite reductases.

In addition to the 50-kDa (alpha) and 40-kDa (beta) subunits, an 11-kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted alpha 2 beta 2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 11-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxamicus (Monticello), D. gigas and D. desulfuricans ATCC 27774. We attribute an alpha 2 beta 2 gamma 2 subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the alpha, beta and gamma subunits are reported.