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 µmol/min per mg of protein. The
activity increased to 40 µmol upon the addition of soybean
phospholipids,
n-octyl-b-D-glucopyranoside,
or its thio derivative to the assay system. Apparent Km
values for NO and phenazine methosulfate were 60 and 2 µM,
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.