Two conserved glutamates in the bacterial nitric oxide reductase are essential for activity but not assembley of the enzyme

Butland, G., Spiro, S., Watmough, N.J. and Richardson, D.J. ORCID: (2001) Two conserved glutamates in the bacterial nitric oxide reductase are essential for activity but not assembley of the enzyme. Journal of Bacteriology, 183 (1). pp. 189-199. ISSN 0021-9193

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The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an FeB–heme-Fe dinuclear catalytic center rather than a CuB–heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues inParacoccus denitrificans NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the cycA promoter to drive aerobic expression of NOR in P. denitrificans and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome bc complex inEscherichia coli. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in P. denitrificans and E. coli. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.

Item Type: Article
Faculty \ School: Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Molecular Microbiology
Faculty of Science > Research Groups > Organisms and the Environment
Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
Depositing User: EPrints Services
Date Deposited: 01 Oct 2010 13:36
Last Modified: 16 May 2023 12:30
DOI: 10.1128/JB.183.1.189-199.2001

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