Journal
BIOCHEMICAL JOURNAL
Volume 401, Issue -, Pages 111-119Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20060856
Keywords
cupredoxin; denitrification; haem-copper oxidase; nitric oxide reductase; nitrous oxide; proton movement
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Funding
- Biotechnology and Biological Sciences Research Council [BB/C007719/1] Funding Source: Medline
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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 Glu(122) or Glu(125) is substituted with a glutamine residue, but only replacement of Glu(122) with ail 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.
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