UNLABELLED: FerB is a flavin mononucleotide (FMN)-containing NAD(P)H: acceptor oxidoreductase of unknown function that is found in the cytoplasm of the bacterium Paracoccus denitrificans. Based on measurements of fluorescence anisotropy, we report here that recombinant FerB readily binds to artificial membrane vesicles. If ubiquinone is incorporated into the membrane, FerB catalyzes its conversion to ubihydroquinone, which may be followed fluorimetrically (with ferricyanide and pyranine entrapped inside the liposomes) or by HPLC. FerB also reduces exogenously added superoxide or superoxide that has been enzymatically generated by the xanthine/xanthine oxidase system or P. denitrificans membrane vesicles. In whole cells, deficiency of FerB increases sensitivity to methyl viologen, as indicated by a lower growth rate and increased production of reactive aldehydes (by-products of lipid oxidation). Taken together, these data support a role for FerB in protection of cells against lipid peroxidation-mediated oxidative stress, and suggest that FerB is a prokaryotic counterpart of mammalian NAD(P)H: quinone oxidoreductase 1.
- MeSH
- antioxidancia chemie metabolismus MeSH
- flavoproteiny chemie metabolismus MeSH
- kinetika MeSH
- membránové proteiny chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres * MeSH
- Paracoccus denitrificans enzymologie MeSH
- superoxidy metabolismus MeSH
- ubichinon metabolismus MeSH
- xanthin metabolismus MeSH
- xanthinoxidasa metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aminokyseliny chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- biokatalýza MeSH
- difrakce rentgenového záření MeSH
- flavinmononukleotid chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- flavoproteiny chemie genetika metabolismus MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- mutageneze cílená MeSH
- NADH, NADPH oxidoreduktasy chemie klasifikace metabolismus MeSH
- NADP chemie metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans enzymologie genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- terciární struktura proteinů * MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
