Electron transport to nitrogenase in <i>Azotobacter chroococcum</i>: Azotobacter flavodoxin hydroquinone as an electron donor

Yates, M. G.

doi:10.1016/0014-5793(72)80410-1

Cited by 69 publications

(60 citation statements)

References 22 publications

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“…Flavodoxin exists in three redox states, the oxidized, semiquinone, and fully reduced (hydroquinone) forms. Only the hydroquinone/semiquinone couple, with an E 0 Ј of about Ϫ500 mV, can transfer electrons to nitrogenase in cyanobacteria (32) and in Azotobacter (235). Reduction of flavodoxin to the fully reduced state does not occur effectively using NAD(P)H [E 0 Ј of NA(P)H/NAD(P) ϩ ϭ Ϫ320 mV], but it can proceed via photosystem I or from pyruvate (E 0 Ј for the pyruvate cleavage ϳ Ϫ500 mV).…”

Section: (Escherichia Coli)mentioning

confidence: 99%

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Bothe

Schmitz²,

Yates

et al. 2010

Microbiol Mol Biol Rev

358

222

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SUMMARY This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N2 fixation and/or H2 formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H2 as a source of combustible energy. To enhance the rates of H2 production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H2 formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.

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Section: (Escherichia Coli)mentioning

confidence: 99%

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Bothe

Schmitz²,

Yates

et al. 2010

Microbiol Mol Biol Rev

358

222

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“…Although this protein contains only one flavin mononucleotide prosthetic group, like AvFdI it is capable of transferring two low-potential electrons at -270 and -460 mV (25). Only the lower-potential couple is believed to be involved in N2 fixation (9,24). Recently, the gene encoding AvFld has been shown to share sequence identity with the nifF gene from Klebsiella pneumoniae and to reside in an analogous position in the nif cluster of the A. vinelandii chromosome (4).…”

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confidence: 99%

“…Recently, the gene encoding AvFld has been shown to share sequence identity with the nifF gene from Klebsiella pneumoniae and to reside in an analogous position in the nif cluster of the A. vinelandii chromosome (4). Because it has been definitively demonstrated that the niJF gene product is the immediate electron donor to nitrogenase in K. pneumoniae (17), the analogous protein is likely to have the same function in Azotobacter species (4,24). In support of this idea is the observation that an Azotobacter mutant unable to synthesize normal Fld had somewhat reduced in vivo nitrogenase activity (4).…”

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confidence: 99%

“…Next, we suggest that FdI normally reduces Fld, a proposal consistent with both the relative reduction potentials of AvFdI and Fld (1,25) and biochemical evidence that they are both involved in N2 fixation in vivo (26). It is likely that Fld serves as only a one-electron acceptor in vivo (24), and theoretically, FdI either could serve as a one-electron donor, using the other electron for some other purpose, or could reduce two different molecules of Fld. Once reduced, Fld could serve as an electron donor for nitrogenase (4,9,24) Conditions are identical to those described previously (12).…”

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confidence: 99%

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Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I

et al. 1989

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Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (niff) and ferredoxin I (f&dA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.The structure of Azotobacter vinelandii ferredoxin I (AvFdI) (nomenclature of Yoch and Arnon [26]) has recently been reexamined by X-ray crystallography (19,20). This unusually large bacterial ferredoxin contains one [4Fe-4S]2+cluster and one [3Fe-4S]f cluster in its air-oxidized state (5). The 3Fe center undergoes pH-dependent one-electron reduction at --420 mV versus standard hydrogen electrode (11,13,22), while studies of the analogous Azotobacter chroococcum FdI show that the 4Fe center has one of the lowest potentials (--645 mV) so far reported for a biological [Fe-S] cluster (1).Despite the wealth of available information on the structure and reactivity of this protein, its biological function remains obscure. The only function so far proposed for AvFdI is as an electron donor to nitrogenase. This proposal is based on the observation that a DEAE-cellulose-treated extract of A. vinelandii could support nitrogenase activity from NADPH but only after the addition of AvFdI and spinach NADP-ferredoxin oxidoreductase (3). This proposal is also consistent with the observation that AvFdI can serve as a direct electron donor to nitrogenase in vitro (26). To better understand the function of this protein, the gene encoding AvFdI (fdxA) has recently been cloned and sequenced and a mutant has been constructed which cannot synthesize FdI (12). These experiments showed thatfdxA is not a nif gene, that AvFdI is constitutively expressed, and that elimination of FdI in vivo has no effect on the growth rate of A. vinelandii under N2-fixing c...

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“…flavodoxin from Anacystis nidulans (phytoflavin) was shown to be more effective in this reaction than Azotobacter flavodoxin [6]. Yates provided more direct evidence by using purified nitrogenase components and dithionite-reduced Azotobacter flavodoxin [7]. The reduced flavodoxin was reoxidized by Azotohacter nitrogenase, in the presence of ATP, to the semiquinone, and the reaction occurred at a reasonable rate.…”

mentioning

confidence: 99%

Regulation of Nitrogen Fixation by Fe‐S Protein II in Azotobacter vinelandii

Scherings¹,

Haaker²,

Veeger³

1977

European Journal of Biochemistry

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1. Several low-potential electron carriers from different sources can be photoreduced by the system 3,l O-dimethyl-5-deazaisoalloxazine/N-tris(hydroxymethyl)methylglycine. The carriers studied were flavodoxin, ferredoxin 1 and iron-sulfur protein I1 from Azotohacter vinelundii and the flavodoxins from Desulfovibrio vulgaris and Peptostreptococcus elsdenii.2. Electron transport to A . vinelandii nitrogenase was studied, employing different preparations of the enzyme: a crude complex; a complex reconstituted from the 0.27 M and 0.38 M NaCl fractions after DEAE-cellulose chromatography; a complex reconstituted from the 0.27 M and 0.38 M NaCl fraction plus iron-sulfur protein I1 purified from the 0.15 M NaCl fraction. Of all photoreduced carriers tested, only flavodoxin hydroquinone from A . vinelundii catalyzes significant electron transport to these complexes.3. The time course of oxidation of substrate-amounts of A . vinelandii flavodoxin hydroquinone by catalytic amounts of crude nitrogenase complex shows three characteristic phases : an initial lag phase (l), a phase with constant rate over a range of redox potentials (2) and a final phase with rapidly declining rate (3). It was shown that the Fe-S protein I1 is responsible for the lag phase; the potential where phase 2 changes into phase 3 is at a higher value in the presence of Fe-S protein 11. Pre-reduction of the enzyme photochemically abolishes phase 1 and causes phase 2 to proceed at a higher rate. The rate in phase 2 can be enhanced also by lowering the 'starting potential' of the flavodoxin hydroquinone/semiquinone couple. 4. A . vinelandii flavodoxin shuttles between its hydroquinone and semiquinone forms during steady-state electron transfer. Over 90 % of the reducing equivalents is recovered in ethylene formed from acetylene. The concentrations of flavodoxin hydroquinone to give half-maximum rate in the acetylene reduction assay is 3 -6 pM. A scheme is proposed for the regulation of electron donation to nitrogenase.It is well established that pyruvate is a main source of reducing equivalents for nitrogenase in many aerobic nitrogen-fixing bacteria, and that electrons are usually transferred from pyruvate dehydrogenase via ferredoxin [l]. Benemann et al. [2] as well as Yoch and Arnon [3] considered ferredoxin a possible candidate as electron donor for nitrogen fixation in Azotohacter, but attempts to demonstrate pyruvate-ferredoxin oxidoreductase activity in extracts have given equivocal results [4].The role of flavodoxin in aerobic nitrogen-fixing organisms is also not certain. As in aerobic organisms it might substitute for ferredoxin under conditions of iron-deficiency. Benemann et al. [2,5] were the first to postulate a role of flavodoxin in nitrogen fixation of Azotobacter. They showed that flavodoxin could couple the reducing power of chloroplast photosystem I to nitrogenase in a cell-free extract. However, the activity obtained amounted to only a fraction of the activity with dithionite as electron donor. In addition, the specificity of the re...

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Electron transport to nitrogenase in Azotobacter chroococcum: Azotobacter flavodoxin hydroquinone as an electron donor

Cited by 69 publications

References 22 publications

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I

Regulation of Nitrogen Fixation by Fe‐S Protein II in Azotobacter vinelandii

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