The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from <i>Azotobacter chroococcum</i> and the effects of inhibitors

Kelly, Michael T.

doi:10.1042/bj1070001

Cited by 47 publications

(15 citation statements)

References 19 publications

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“…M.S.3 mass spectrometer either locally or by Dr C. W. Crane Acetylene and isocyanide reduction. All unequivocally nitrogen-king organisms SO far examined reduce acetylene to ethylene (Dilworth, 1966;Schollhorn & Burris, 1967) and methyl isocyanide to methane (Kelly, 1968). These reactions provide very sensitive tests for the presence of the nitrogenase system since the products can be detected by gas chromatography.…”

Section: Organismsmentioning

confidence: 99%

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

Hill

Postgate

1969

Journal of General Microbiology

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Section: Organismsmentioning

confidence: 99%

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

Hill

Postgate

1969

Journal of General Microbiology

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“…and for the strict anaerobe, Clostridium pasteurianum have been reported by several groups of workers (1)(2)(3)(4), few investigations have been made with the enzyme systems from the facultative anaerobes, Klebsiella pneumoniae and Bacillus polymyxa. Mahl and Wilson (5) reported the first cell-free extract that fixed N2 from K.…”

mentioning

confidence: 99%

Kinetic Studies on Klebsiella pneumoniae Nitrogenase

Parejko

Wilson

1971

Proc. Natl. Acad. Sci. U.S.A.

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Purified cell-free extracts of Klebsiella pneumoniae reduce N2, N3-, CN -, or pneumoniae, but attempts to estimate the Km for N2 reduction were unsuccessful, owing to the weak activity of the preparations. We have now obtained much more active preparations (6) that allow kinetic studies to be made not only for the reduction of N2, but also for the alternate substrates, cyanide, azide, and acetylene. MATERIALS AND METHODS Preparation of cell-free extractsCultures of K. pneumoniae M5al were grown and harvested as described, (5, 6) with the exception that 25 mM Tris * HC1 (pH 7.4) (7 ml/liter of culture; Sigma Chemical Co., St. Louis, Mo.), was used instead of phosphate buffer. Although cell-free extracts were routinely prepared by the use of a French pressure cell, active extracts have also been prepared by the use of lysozyme, deoxyribonuclease, and a detergent.Cell-free extracts were used fresh or stored frozen. The method of storage was to freeze the extract in a capped serum bottle that had been placed in a Brewer jar that had been evacuated, flushed, and filled with N2.Purification of nitrogenaseThe crude extract (30-40 mg of protein/ml) was heated at 650C for 5 min in 15-ml centrifuge tubes sealed with rubber stoppers through which N2 gas could be flushed. After cooling to 10'C in an ice bath, the extract was centrifuged at 25,000 X g for 30 min, resulting in a supernatant solution containing 14-16 mg of protein/ml. The heat treatment resulted in about a 2-fold increase in specific activity (N2 reduction), but with a loss of about 40% of the total units of activity.Some of the protein removed by routine heat-treatment includes the classical hydrogenase system of this bacterium, which is responsible for H2 evolution from reduced methyl viologen or pyruvate; however, the ATP-dependent hydrogenase activity (associated with nitrogenase) increased concomitantly with the N2-reducing activity. The method of anaerobic DEAE-cellulose chromatography was that described by Detroy et al. (7); we used 2.5 X 10 cm columns of DEAE-cellulose (Sigma), with an exchange capacity of 0.9-1.10 meq/g. AssaysThe methods for assay, essentially those routinely used in this field, have been described (4,5,8,9). For the Nrreduction assays, an atmosphere of N2 was used in the duplicate experimental bottles and an atmosphere of He was used for the controls. NaN3 (Eastman Organic Chemicals, Rochester, N.Y.) and KCN (City Chemical Co., New York) were supplied at 10 mM concentrations under a He gas phase. The ammonia produced from either of these substrates was distilled by microdiffusion and quantitatively analyzed by nesslerization. The acetylene reduction assay consumed 0.3 cm3 of C2H2 (The Matheson Co., Inc., East Rutherford, N.J.), which was injected into the He gas phase of duplicate 6-ml serum bottles. Controls for each of these assays (azide, cyanide, and acetylene) were performed by the use of complete reaction mixtures without substrate. The reactions for N2 and N3-reduction were incubated at 30'C, while those for CN -a...

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“…Ethylene, like ammonia, does not complex strongly with N2ase, being neither inhibitory to N2 fixation nor reduced by N2ase (26,53,54). The affinity of C2H2 for N2ase is similar to that of N2 for N2ase (26,50,53,54,57). The rate of reduction is four times that of N2 and the rate of electron consumption is about 1.3 times that of N2 (58).…”

Section: Figure 3 Classes Of Substrates Reduced By N2asementioning

confidence: 96%

“…It is not known whether HCN or CN" is the actual substrate, since both species are present at reaction pH. The affinity for cyanide based on equilibrium concentration of HCN is intermediate between that for N2 or C2H2 and N20 or N3", while the affinity based on CN" concentration is substantially greater (28,46,50). The rate of electron consumption is less than that seen in N2 reduction, which may indicate partial inhibition of N2ase by cyanide and/or the generation of undetermined products.…”

Section: Figure 3 Classes Of Substrates Reduced By N2asementioning

confidence: 96%

“…No enzyme-free intermediates have been detected (42,43,44) and, on the basis of 2-, 4-, 6-, 8-, 10-, 12-, and 14-electron addition products found in other N2ase-catalyzed reactions (Figure 3), enzyme-bound diimide and hydrazine are the obvious, but not yet detected, intermediates. The N2 concentration for half maximal rate is about 0.1 mM (15,29,45,46,47,48,49,50).…”

Section: Figure 3 Classes Of Substrates Reduced By N2asementioning

confidence: 99%

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The Biochemistry of N₂ Fixation

Hardy

Burns

Parshall

1971

Advances in Chemistry

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The nature of the N2ase reaction is described with respect to electron donation, energy requirement, and reduction characteristics, with particular analysis of the seven classes of substrates reducible by N2ase, a complex of a Mo-Fe and Fe protein. Chemical and physical characteristics of Fe protein and crystalline Mo-Fe protein are summarized. The two-site mechanism of electron activation and substrate complexation is further developed. Reduction may occur at a biological dinuclear site of Mo and Fe in which N2 is reduced to NH3 via enzyme-bound diimide and hydrazine. Unsolved problems of electron donors, ATP function, H2 evolution and electron donation, substrate reduction, N2ase characteristics and mechanism, and metal roles are tabu lated. Potential utilities of N2 fixation research include in creased protein production and new chemistry of nitrogen.'"phe facile reduction of N2 to NH3 by the natural catalyst, nitrogenase, A is not understood and has never been equalled with synthetic cata lysts. Developments of the past decade may change this. A renaissance in N2 fixation research in both biochemistry and inorganic chemistry is providing the first opportunity for bioinorganic interaction between these previously independent approaches to catalytic N2 reduction. Remark able progress-from crude extracts to crystalline protein and from a single N2ase substrate to recognition of seven classes of substrates-in the elu cidation of the complex biochemistry of N2 fixation has been made during the past decade ( Figure 1) (1,2,3 , 4, 5, 6, 7, 8, 9, 10, 11). Moreover, advances in the inorganic chemistry of N2-fixation-synthesis of transition metal complexes of N2 and reduction of N2 under ambient conditionsare yielding the first examples of bioinorganic models.We will summarize the status of the biochemistry of N2 fixation, including nitrogenase (N2ase) reactions and characteristics, with exten-219

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The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from Azotobacter chroococcum and the effects of inhibitors

Cited by 47 publications

References 19 publications

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

Kinetic Studies on Klebsiella pneumoniae Nitrogenase

The Biochemistry of N₂ Fixation

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The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from Azotobacter chroococcum and the effects of inhibitors

Cited by 47 publications

References 19 publications

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

Kinetic Studies on Klebsiella pneumoniae Nitrogenase

The Biochemistry of N2 Fixation

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The Biochemistry of N₂ Fixation