NDLI: Two Conserved Glutamates in the Bacterial Nitric Oxide Reductase Are Essential for Activity but Not Assembly of the Enzyme

Content Provider	PubMed Central
Author	Butland, Gareth Spiro, Stephen Watmough, Nicholas J. Richardson, David J.
Copyright Year	2001
Abstract	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 in Paracoccus 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 in Escherichia 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.
Related Links	http://dx.doi.org/10.1128/JB.183.1.189-199.2001
Starting Page	189
File Format	PDF
ISSN	10985530
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	1
Volume Number	183
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-01-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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