A new assay for nitric oxide reductase reveals two conserved glutamate residues form the entrance to a proton-conducting channel in the bacterial enzyme.
A specific amperometric assay was developed for the membrane-bound NOR [NO (nitric oxide) reductase] from the model denitrifying bacterium Paracoccus denitrificans using its natural electron donor, pseudoazurin, as a co-substrate. The method allows the rapid and specific assay of NO reduction catalysed by recombinant NOR expressed in the cytoplasmic membranes of Escherichia coli. The effect on enzyme activity of substituting alanine, aspartate or glutamine for two highly conserved glutamate residues, which lie in a periplasmic facing loop between transmembrane helices III and IV in the catalytic subunit of NOR, was determined using this method. Three of the substitutions (E122A, E125A and E125D) lead to an almost complete loss of NOR activity. Some activity is retained when either Glu122 or Glu125 is substituted with a glutamine residue, but only replacement of Glu122 with an aspartate residue retains a high level of activity. These results are interpreted in terms of these residues forming the mouth of a channel that conducts substrate protons to the active site of NOR during turnover. This channel is also likely to be that responsible in the coupling of proton movement to electron transfer during the oxidation of fully reduced NOR with oxygen [U. Flock, N. J. Watmough and P. Adelroth (2005) Biochemistry 44, 10711-10719].
Thorndycroft
Faye H
FH
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norfolk NR4 7TJ, UK.
Butland
Gareth
G
Richardson
David J
DJ
Watmough
Nicholas J
NJ
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D009609:Nitrous Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000254:growth & development; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide; D018698:Glutamic Acid; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D009609:Nitrous Oxide