<i>Klebsiella pneumoniae</i> nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide

Lowe, David J.; Fisher, Karl; Thorneley, R. N. F.

doi:10.1042/bj2720621

Cited by 64 publications

(86 citation statements)

References 18 publications

(15 reference statements)

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“…In contrast, C 2 H 2 is proposed to bind reversibly to species E 1 and E 2 of the MoFe protein, which are also involved in H 2 formation. This possibility is consistent with C 2 H 2 being a more effective competitor of H 2 evolution than is N 2 (Lowe et al, 1990). Finally, the fact that N 2 must bind at a more reduced level than does C 2 H 2 also explains the observed nonreciprocity of kinetic patterns when both are present.…”

supporting

confidence: 65%

Evidence for Multiple Substrate-Reduction Sites and Distinct Inhibitor-Binding Sites from an Altered Azotobacter vinelandii Nitrogenase MoFe Protein

1997

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The arginine-277 residue of the alpha-subunit of the nitrogenase MoFe protein was targeted for substitution because it is (i) a close neighbor of alpha-cysteine-275, which is one of only two residues anchoring the FeMo cofactor to the polypeptide, and (ii) a component of a potential channel for entry/exit of substrates/products and for accepting FeMo cofactor during MoFe-protein maturation. Several of the eight mutant strains constructed were capable of good diazotrophic growth and also contained FeMo cofactor as indicated by its biologically unique S = 3/2 EPR spectrum. These observations indicate that the positively charged alpha-arginine-277 residue is not required for acceptance of the negatively charged FeMo cofactor by the separately synthesized, cofactor-deficient, apo-MoFe protein. The wide range of nitrogen-fixation phenotypes shown by these mutant strains generally correlated well with their C2H2- and proton-reduction activities, which range from 5 to 65% of wild-type activity. One notable exception is the histidine-substituted strain, DJ788 (alpha-277His). This strain, although unable to fix N2 and grow diazotrophically, elaborates an altered alpha-277His MoFe protein that catalyzes the reduction of the alternative substrates, C2H2, HCN, HN3, and protons. These observations are best explained if multiple redox levels are available to the MoFe protein but the alpha-277His MoFe protein is incapable of reaching the more-reduced redox levels required for nitrogen fixation. Under nonsaturating CO concentrations, the alpha-277His MoFe-protein-catalyzed reduction of C2H2 showed sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity among two C2H4-evolving sites and indicates the presence of three sites, which can be simultaneously occupied, on the MoFe protein. Similar kinetics were not observed for alpha-277His MoFe-protein-catalyzed reduction of either HCN or HN3 with nonsaturating CO levels, indicating that these substrates are unlikely to share common binding sites with C2H2. Further, CN- did not induce cooperativity in C2H2 reduction and, therefore, CO and CN- are unlikely to share a common binding site. These changed substrate specificities, reinforced by changes in the FeMo-cofactor-derived S = 3/2 EPR spectrum, clearly indicate the importance of the alpha-277 residue in catalysis and the delicate control exerted on the properties of bound FeMo cofactor by its polypeptide environment.

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supporting

confidence: 65%

Evidence for Multiple Substrate-Reduction Sites and Distinct Inhibitor-Binding Sites from an Altered Azotobacter vinelandii Nitrogenase MoFe Protein

1997

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“…We have defined a kinetic scheme that is the generally accepted basis for the understanding of the mechanism of N2 and HI reduction by nitrogenase (Orme-Johnson, 1992), in a series of five papers (Thorneley and Lowe, 1983; Lowe and Thorneley, 1984a,b;Thorneley and Lowe, 1984a,b). This has subsequently been extended to include a description of the reduction of C2H2 by the enzyme (Lowe et al, 1990). The mechanism is described in terms of the Fe-protein (Kp2) cycle and the MoFe-protein (Kpl) cycle.…”

Section: Introductionmentioning

confidence: 99%

“…H2 can be evolved from E2, E3 and E4 (Scheme 1) independently of N2 binding. C2H2 binds at E1 and E2 and C2H4 is evolved from E3 (Lowe et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?

Lowe

Fisher

Thorneley

1993

Biochemical Journal

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The pre-steady-state absorbance changes that occur during the first 0.6 s of reaction of the nitrogenase of Klebsiella pneumoniae can be simulated by associating redox changes with the different states of the MoFe protein described by our published kinetic model for nitrogenase [Lowe and Thorneley (1984) Biochem. J. 224, 877-886]. When the substrate is changed, from H+ to C2H2 (acetylene) or N2, or the nitrogenase component protein ratio is altered, these pre-steady-state absorbance changes are affected in a manner that is quantitatively predicted by our model. The results, together with parallel e.p.r. studies, are interpreted as showing that the P-clusters become oxidized when the MoFe protein is in the state where bound N2 is irreversibly committed to being reduced and is protonated to the hydrazido(2-) level.

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“…It is also known that when N, is reduced, the P clusters become oxidised [21]. There is no direct evidence of an additional ligand being bound to molybdenum of nitrogenase during the reduction process, though early data [22] suggesting light-atom ligands to molybdenum are substantiated by the X-ray structure.…”

Section: Mh3 -mentioning

confidence: 56%

The Mechanism of Dinitrogen Reduction by Molybdenum Nitrogenases

Leigh

1995

European Journal of Biochemistry

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The recent descriptions of the crystal structure of the molybdenum-iron protein and the iron protein of Azotobacter vinelandii nitrogenase [ 11 and of Clostridium pasteurianum nitrogenase [2] have been exciting and frustrating, exciting because the structures have been sought for more than 30 years, and frustrating because the structures do not obviously reveal the active site, nor do they really imply a specific active site.This has not prevented speculation and calculation to determine how nitrogenase actually works [3, 41. Simple speculation is easier, but should always attempt to remain consistent with the great deal we do know about the process of biological nitrogen fixation. Calculation is more dangerous. In the past, theoreticians have paid relatively little attention to chemical facts, and relied upon their imagination. The results of the calculations may indeed indicate a preferred pathway among those considered. However, there is no guarantee that the pathways considered include the one that nitrogenase uses. We are limited by our imagination and our knowledge. Consequently, it behoves us, when considering a mechanism, to use all the data we can. This short review is designed to do just that.The structure of the metal clusters at the core of the molybdenum-iron protein in both A. vinelandii [l] and C. pasteurianum [2] are shown in Fig. 1. The smaller eight-iron system, which is believed to act as an electron-transfer agent to the ironmolybdenum cofactor/cluster (FeMoco), is not believed to be involved directly with binding and reducing dinitrogen. There are at least three genetically distinct kinds of nitrogenase [5] based on iron and molybdenum, iron and vanadium, and iron and no other evident metal. These three nitrogenases, with a common iron constituent, have belled the speculation that iron, and not molybdenum or vanadium, is at the active site of nitrogenase [6]. Currently, there is no definitive answer to this problem, but this has not curbed inventive minds.As an example, there have been at least three sets of calculations performed investigating the ways in which dinitrogen might bind to the seven-iron residue of the FeMoco. One 131 was a bit previous, in that the residue initially not identified, but labelled Y, was postulated to be N,. Now, the consensus appears to be that Y is actually sulfur [7]. Another two calculations discuss N, binding to iron clusters [4, 81.

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Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide

Cited by 64 publications

References 18 publications

Evidence for Multiple Substrate-Reduction Sites and Distinct Inhibitor-Binding Sites from an Altered Azotobacter vinelandii Nitrogenase MoFe Protein

Evidence for Multiple Substrate-Reduction Sites and Distinct Inhibitor-Binding Sites from an Altered Azotobacter vinelandii Nitrogenase MoFe Protein

Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?

The Mechanism of Dinitrogen Reduction by Molybdenum Nitrogenases

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