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A new assay for nitric oxide reductase reveals two conserved glutamate residues form the entrance to a proton-conducting channel in the bacterial enzyme. | LitMetric

AI Article Synopsis

  • A specific amperometric assay was developed to measure the nitric oxide reductase (NOR) enzyme from the bacterium Paracoccus denitrificans, utilizing pseudoazurin as a natural electron donor.
  • The assay enabled examination of how substitutions in two conserved glutamate residues (E122 and E125) in the enzyme's structure affected its activity, with some mutations leading to significant loss of function.
  • The findings suggest that these glutamate residues are crucial for creating a channel that allows substrate protons to access the active site of NOR, which also facilitates electron transfer during reactions with oxygen.

Article Abstract

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].

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698692PMC
http://dx.doi.org/10.1042/BJ20060856DOI Listing

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