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J Bacteriol. Jun 1989; 171(6): 3288–3297.
PMCID: PMC210048

Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri.

Abstract

Nitric oxide (NO) reductase was solubilized by Triton X-100 from the membrane fraction of Pseudomonas stutzeri ZoBell and purified 100-fold to apparent electrophoretic homogeneity. The enzyme consisted of two polypeptides of Mr 38,000 and 17,000 associated with heme b and heme c, respectively. Absorption maxima of the reduced complex were at 420.5, 522.5, and 552.5 nm, with a shoulder at 560 nm. The electron paramagnetic resonance spectrum was characteristic of high- and low-spin ferric heme proteins; no signals typical for iron-sulfur proteins were found. Nitric oxide reductase stoichiometrically transformed NO to nitrous oxide in an ascorbate-phenazine methosulfate-dependent reaction with a specific activity of 11.8 mumols/min per mg of protein. The activity increased to 40 mumols upon the addition of soybean phospholipids, n-octyl-beta-D-glucopyranoside, or its thio derivative to the assay system. Apparent Km values for NO and phenazine methosulfate were 60 and 2 microM, respectively. The pH optimum of the reaction was at 4.8. Cytochrome co was purified from P. stutzeri to permit its distinction from NO reductase. Spectrophotometric binding assays and other criteria also differentiated NO reductase from the respiratory cytochrome bc1 complex.

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