These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Access to the active site of periplasmic nitrate reductase: insights from site-directed mutagenesis and zinc inhibition studies.
    Author: Dementin S, Arnoux P, Frangioni B, Grosse S, Léger C, Burlat B, Guigliarelli B, Sabaty M, Pignol D.
    Journal: Biochemistry; 2007 Aug 28; 46(34):9713-21. PubMed ID: 17676770.
    Abstract:
    The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding: the Vm measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the Km for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (Ki' = 1 microM). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.
    [Abstract] [Full Text] [Related] [New Search]