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The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.

ABSTRACT: FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.

SUBMITTER: Sedlacek V

PROVIDER: S-EPMC4015959 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.

Sedláček Vojtěch V Klumpler Tomáš T Marek Jaromír J Kučera Igor I

PloS one 20140509 5

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein c ...[more]

PMID: 24817153

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Project description:In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nosZ gene, is the only biological pathway for N2O consumption. In this study, we successfully isolated a strain of denitrifying Paracoccus denitrificans R-1 from sewage treatment plant sludge. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10 ± 0.11 × 10-9 µmol·h-1·cell-1 under anaerobic condition in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is related to the energy generation process. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, nosL, and nosZ, which was identified as that in other strains in clade I. Respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. These findings suggest that modular N2O reduction of P. denitrificans R-1 is linked to the electron transport and energy conservation, and dissimilatory N2O reduction is a form of microbial anaerobic respiration.ImportanceNitrous oxide (N2O) is a potent greenhouse gas and contributor to ozone layer destruction, and atmospheric N2O has increased steadily over the past century due to human activities. The release of N2O from fixed N is almost entirely controlled by microbial N2O reductase activities. Here, we investigated the ability to obtain energy for the growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. The modular N2O reduction process of denitrifying microorganism not only can consume N2O produced by itself but also can consume the external N2O generated from biological or abiotic pathways under suitable condition, which should be critical for controlling the release of N2O from ecosystems into the atmosphere.

Dataset Information

The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.

Publications

The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.

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