Comparative characterization of H 2 production by the conventional Mo nitrogenase and the alternative "iron-only" nitrogenase of Rhodobacter capsulatus hup - mutants

Krahn, Eugen; Schneider, Klaus; Müller, Achim

doi:10.1007/s002530050818

Cited by 36 publications

(27 citation statements)

References 13 publications

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“…The Fe nitrogenase exhibited an extraordinary high H,-evolving activity. With intact cells and also with the in vitro system, at Rc2/Rcl ratios as they exist in the cell (2: l), the Mo nitrogenase was only slightly superior (see the data of Table 2 and also [38], which contains a more detailed characterization of the itz vivo H, production by R. cupsulatus wild-type and mutant cells). When the isolated components at a 40-fold molar excess of component 2 were used, then the Fe nitrogenase showed higher H, production rates than the Mo nitrogenase.…”

Section: Resultsmentioning

confidence: 99%

“…This characterization included data on subunit structure and some basic catalytical and EPR-spectroscopic properties [5], on the formation of a FeFe protein/FeMo cofactor hybrid enzyme [21], and on the in vivo production of molecular hydrogen [38]. A more comprehensive description and discussion of the properties of this enzyme system including a detailed comparison with the Mo-containing nitrogenase, has so far been lacking and is presented in this article.…”

Section: Discussionmentioning

confidence: 99%

“…This conceined not only O2 sensitivity [38], but also lability towards dilution, freezinghhawing [ 101, and excess concentrations of salts and metal ions [ S ] . The only possibility to obtain an enzyme system with good activity in the purified state despite this instability was to develop a very rapid purification procedure that makes time-consuming, protein-diluting chromatography steps (e.g.…”

Section: Discussionmentioning

confidence: 99%

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Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

Schneider

Gollan

Dröttboom

et al. 1997

European Journal of Biochemistry

125

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The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (AnifhTDK, AmodABCD strain) and purified in a one-day procedure that included only one columnchromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, RclFe), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, R c~~' ) . The highest specific activities, which were achieved at an R c~~V R C~~~ molar ratio of 40: 1, were 260 (C,H, from C,H,), 350 (NH, formation), and 2400 (H, evolution) nmol product formed . min-' . mg protein-'.The purified FeFe protein contained 26 -t 4 Fe atoms ; it did not contain Mo, V, or any other heterometal atom.The most significant catalytic property of the iron-only nitrogenase is its high H,-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N, reduction, the activity ratios (mol N, reduced/mol H, produced) obtained were 1 : 1 (molybdenum nitrogenase) and 1 :7.5 (iron nitrogenase). The Rcl" protein has only a very low affinity for C,H,. The K,, value determined (12.5 kPa), was about ninefold higher than the K,, for RclM" (1.4 kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5.In complementation experiments, component 1 proteins coupled very poorly with the 'wrong' component 2. RclF', if complemented with R c~~" , showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rcl'" and Rc lM" are immunologically not related.The most active RclFe samples appeared to be EPR-silent in the Na,S,O,-reduced state. However, on partial oxidation with K,[Fe(CN),] or thionine several signals occurred. The most significant signal appears to be the one at g = 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and + 150 mV. Another novel EPR signal (g = 1.96, 1.92, 1.77) occurred on further reduction of RclF" by using turnover conditions in the presence of a substrate (N,, C,H,, H+).Keywords: nitrogenase ; iron protein ; cofactor; EPR; Rhodobacter capsulatus.Three genetically distinct types of nitrogenase systems (ng vnJ unf, have so far been proved to exist in nature. The most widespread and intensively characterized system is the classical Mo-containing nitrogenase (nif system) found in all diazotrophs [l, 21. During the last few years, two types of alternative, Moindependent nitrogenases have been discovered and described. One is a vanadium-containing nitrogenase (vnf system) (for review see [3]) and the other enzyme lacks both Mo and V (anf system) and has been tentatively designated as Fe nitrogenase [4][5][6]. Although there is much circumstantial and...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

Schneider

Gollan

Dröttboom

et al. 1997

European Journal of Biochemistry

125

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“…NifA is the transcriptional regulator of the nitrogenase regulon, which includes genes encoding the nitrogenase complex, nifHDK, along with all the other genes whose products are necessary for its assembly, maturation and activation (Buchanan-Wollaston et al, 1981a, b;Dixon & Kahn, 2004;Dixon et al, 1977;Roberts et al, 1978). Numerous modifications in genes encoding proteins in this cascade have been shown to alter the regulation of nitrogenase-catalysed hydrogen production in NSP bacteria (Barbosa et al, 2001;Franchi et al, 2004;Jahn et al, 1994;Khatipov et al, 1998;Koku et al, 2002;Krahn et al, 1996;Vasilyeva et al, 1999;Willison et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides

et al. 2014

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Previously, the RubisCO-compromised spontaneous adaptive Rhodobacter sphaeroides mutant, strain 16PHC, was shown to derepress the expression of genes that encode the nitrogenase complex under normal repressive conditions. As a result of this adaptation, the active nitrogenase complex restored redox balance, thus allowing strain 16PHC to grow under photoheterotrophic conditions in the absence of an exogenous electron acceptor. A combination of whole genome pyrosequencing and whole genome microarray analyses was employed to identify possible loci responsible for the observed phenotype. Mutations were found in two genes, glnA and nifA, whose products are involved in the regulatory cascade that controls nitrogenase complex gene expression. In addition, a nucleotide reversion within the nifK gene, which encodes a subunit of the nitrogenase complex, was also identified. Subsequent genetic, physiological and biochemical studies revealed alterations that led to derepression of the synthesis of an active nitrogenase complex in strain 16PHC.

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“…While all characterized nitrogenases produce molecular hydrogen as a by-product, the iron-only nitrogenase generates greater amounts of molecular hydrogen for each molecule of N 2 fixed than the molybdenum nitrogenase (4,10). Wild-type A. vinelandii possesses a membrane-bound uptake hydrogenase which reoxidizes hydrogen that nitrogenases produce, recovering the energy rather than allowing the gas to evolve (11)(12)(13)(14).…”

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confidence: 99%

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

Noar

Loveless

Navarro

et al. 2015

Appl Environ Microbiol

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The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.

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Comparative characterization of H 2 production by the conventional Mo nitrogenase and the alternative "iron-only" nitrogenase of Rhodobacter capsulatus hup - mutants

Cited by 36 publications

References 13 publications

Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

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