Affiliation:
1. Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
Abstract
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 Fe
B
–heme-Fe dinuclear catalytic center rather than a Cu
B
–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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
101 articles.
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