<i>Klebsiella pneumoniae</i> nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane

Ashby, G. A.; Dilworth, M. J.; Thorneley, R. N. F.

doi:10.1042/bj2470547

Cited by 44 publications

(44 citation statements)

References 25 publications

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“…It is based on the scheme of Ashby et al (1987) with the addition of intermediatef, which they did not consider, …”

Section: Resultsmentioning

confidence: 99%

“…In the present paper we describe the extension of the mechanism to C2H2 (acetylene) reduction, which is important for two reasons: first because it is commonly used to assay nitrogenase activity both in vivo and in vitro (Koch & Evans, 1966;Hardy et al, 1968), and secondly because it sheds light on the chemistry occurring at the substrate-reduction site. Ashby et al (1987) have shown that C2H4 (ethylene), the product of C2H2 reduction, is itself slowly reduced to C2H6 (ethane) by nitrogenase, although this reaction is inhibited by C2H2. C2H4 was also shown to inhibit the total electron flux through nitrogenase and the hydrolysis of MgATP to the same extent.…”

Section: Introductionmentioning

confidence: 99%

“…272 duction to yield C2H6. Ashby et al (1987) (Burgess, 1985), which suggests that it binds at a different site or at a different oxidation level of the same site.…”

Section: Introductionmentioning

confidence: 99%

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

Lowe

Fisher

Thorneley

1990

Biochemical Journal

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The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S204) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kpl) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kpl. This explanation is also consistent with a pre-steady-state lag phase for C24 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kpl and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKpIC2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme.

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“…It is based on the scheme of Ashby et al (1987) with the addition of intermediatef, which they did not consider, …”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Lowe

Fisher

Thorneley

1990

Biochemical Journal

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“…This reaction was strongly inhibited by acetylene. Moreover, it was confirmed that a direct reduction of acetylene to ethane was not catalyzed (Ashby et al, 1987).…”

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confidence: 92%

Detection of the in vivo incorporation of a metal cluster into a protein

Gollan

Schneider

Müller

et al. 1993

European Journal of Biochemistry

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The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The Mo0,2-concentration causing 50 % repression of the alternative nitrogenase in nijHDK-cells was 6 nM. If MOO:-was added to a growing nijHDK-culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C,H, formation decreased continuously during the duration of the experiment (3 days), the total C,H, production increased about twofold within the first 24 h, whereas the relative yield of C,H, increased from 2 % (C,H$C,H, X 100) in the absence of MOO:-, to a maximal value of 69% in the presence of MOO:-(1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MOO:-concentration in the medium and the longer the incubation time. In the presence of ReO;, WO2-or VO:-, a similar effect did not occur.The 'Mo effect' was not observed in a nijHDK-nijZ-double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nijHDK-nifQ-mutant.Crude extracts from nifHDK-cells cultivated in the presence of MOO:-, also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase.The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of niP-lacZ and nijiV-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MOO:-deficiency.The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.As the metal clusters of nitrogenases ('P' cluster, FeMo cofactor) play a key role in the process of the biological nitrogen fixation, information about their properties and biosynthesis seems to be of significance (Muller and Newton, 1983;Burgess, 1990). The type of cofactor (FeMoco, FeVco, FeFeco) and also the protein environment of this cluster influence the activity and the substrate selectivity of the nitrogenase (Scott et al., 1990;Miiller et al., 1992).The conventional FeMoco-containing nitrogenases have been characterized to reduce acetylene exclusively to ethylene. In a recent work, Ashby and colleagues (1987) demonstrated that the Mo nitrogenase of Klebsiella pneumoniae is

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“…C,H, reduction activity has recently been reported for the Mo-containing nitrogenases from K. pneumoniae and A. chroococcum [33]. In fact, the Xanthobacter nitrogenase was also capable of reducing C,H, to C,H,.…”

Section: Conditionsmentioning

confidence: 90%

The Molybdenum Nitrogenase from Wild-type Xanthobacter autotrophicus Exhibits Properties Reminiscent of Alternative Nitrogenases

Schneider¹,

Müller²,

Krahn³

et al. 1995

Eur J Biochem

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In the presence of molybdate (1 pM) 2-3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen-fixing hydrogen-oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm).The maximal specific activity obtained was 145 nmol ethylene reduced . min-' . mg protein-' (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate-starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X . autotrophicus, a Mo-independent nitrogenase does not exist. However, the molybdate-containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases.The MoFe protein (component 1, Xal) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (111) citrate as electron donor, at high component l/component 2 ratios, and at low, non-saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH-dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S = 3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [ED values of 0.036 (signal I), 0.072 (signal 11) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (111) citrate instead of Na,S,O, as reductant.Keywords. Nitrogenase ; MoFe protein ; FeMo cofactor ; ethane formation; ESR.The 'classical' Mo-containing nitrogenase is a well-characterized N,-reducing enzyme system widespread in microorganisms [1]. It consists of two metalloproteins, component 1 (MoFe protein) and component 2 (Fe protein). The tetrameric MoFe protein (a2P2), on which we focus our main interest in this work, contains two types of unique metal clusters, the so-called 'P' cluster (Fe,S,-,) and the FeMo cofactor (FeMoco, 'M' cluster) which is considered to be the site of substrate binding and reduction. In recent publications by Rees and coworkers [2-41, structural models for both cluster types were proposed, based on crystallographic analyses of the MoFe protein from Azotobacter vinelandii, refined up to 0.22-nm resolution. FeMoco is located in the a subunit and consists of an Fe,S, and a MoFe,S, cluster fragment bridged by probably three p,-sulCorrespondence to A. Muller,

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Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane

Cited by 44 publications

References 25 publications

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

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

Detection of the in vivo incorporation of a metal cluster into a protein

The Molybdenum Nitrogenase from Wild-type Xanthobacter autotrophicus Exhibits Properties Reminiscent of Alternative Nitrogenases

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