The co-immobilization of P450-type nitric oxide reductase and glucose dehydrogenase for the continuous reduction of nitric oxide via cofactor recycling

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. Ädelroth (2005) Biochemistry 44, 10711–10719].

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The Nitric-oxide Reductase fromParacoccus denitrificansUses a Single Specific Proton Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m113.497347 ◽

2013 ◽

Vol 288 (42) ◽

pp. 30626-30635 ◽

Cited By ~ 13

Author(s):

Josy ter Beek ◽

Nils Krause ◽

Joachim Reimann ◽

Peter Lachmann ◽

Pia Ädelroth

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Reductase

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Detergent inhibition of nitric-oxide reductase activity

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(87)90074-4 ◽

1987 ◽

Vol 911 (3) ◽

pp. 334-340 ◽

Cited By ~ 33

Author(s):

J.P. Shapleigh ◽

K.J.P. Davies ◽

W.J. Payne

Keyword(s):

Nitric Oxide ◽

Reductase Activity ◽

Nitric Oxide Reductase

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A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in β- and γ-Proteobacteria

Journal of Bacteriology ◽

10.1128/jb.186.23.7980-7987.2004 ◽

2004 ◽

Vol 186 (23) ◽

pp. 7980-7987 ◽

Cited By ~ 20

Author(s):

Andrea Büsch ◽

Anne Pohlmann ◽

Bärbel Friedrich ◽

Rainer Cramm

Keyword(s):

Nitric Oxide ◽

Transcriptional Activation ◽

Target Genes ◽

Ralstonia Eutropha ◽

Upstream Region ◽

Nitric Oxide Reductase ◽

Wild Type ◽

Genome Database ◽

Nitric Oxide Metabolism ◽

Signaling Domain

ABSTRACT The σ54-dependent regulator NorR activates transcription of target genes in response to nitric oxide (NO) or NO-generating agents. In Ralstonia eutropha H16, NorR activates transcription of the dicistronic norAB operon that encodes NorA, a protein of unknown function, and NorB, a nitric oxide reductase. A constitutively activating NorR derivative (NorR′), in which the N-terminal signaling domain was replaced by MalE, specifically bound to the norAB upstream region as revealed by gel retardation analysis. Within a 73-bp DNA segment protected by MalE-NorR′ in a DNase I footprint assay, three conserved inverted repeats, GGT-(N7)-ACC (where N is any base), that we consider to be NorR-binding boxes were identified. Mutations altering the spacing or the base sequence of these repeats resulted in an 80 to 90% decrease of transcriptional activation by wild-type NorR. Genome database analyses demonstrate that the GT-(N7)-AC core of the inverted repeat is found in several proteobacteria upstream of gene loci encoding proteins of nitric oxide metabolism, including nitric oxide reductase (NorB), flavorubredoxin (NorV), NO dioxygenase (Hmp), and hybrid cluster protein (Hcp).

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