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