The nitric oxide reductase (NOR) from Pseudomonas stutzeri is a cytochrome bc complex which shows on SDS/PAGE two subunits with apparent molecular masses of 17 kDa and 38 kDa. Two other species of  ~45 kDa and 74-78 kDa represent the undissociated enzyme complex and an aggregate of the cytochrome b subunit, respectively. The cytochrome b subunit is highly hydrophobic and results in aberrant electrophoretic mobility. The stability of the enzyme in various detergents and at different pH was investigated. The highest specific activity of 60 µmol NO min^-1 mg^-1 protein was obtained after electrophoresis in the presence of  laurylpropanediol-3-phosphorylcholine ether. Purified NOR contained cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine, the latter as the major component. A phospholipid was required for high catalytic activity with either cardiolipin or phosphatidylglycerol increasing the activity of the enzyme as isolated by a factor of up to 5. Free fatty acids inhibited NOR, with cis-9-octadecenoic acid (oleic acid) showing the most pronounced effect. Certain detergents substituted for the phospholipid requirement of NOR.

The enzyme, as isolated, in 0.1% Triton X-100, 20 mM Tris/HCl pH 8.5, exhibited a complex set of EPR resonances at low magnetic field, with a prominent peak at g 6.34 resulting from Fe(III) high-spin cytochrome b. The second prominent feature arose from a low-spin Fe(III) heme center with strong lines at apparent g values of 3.02 and 2.29, and a broad resonance at g ~ 1.5 which we assigned to the cytochrome c component of the enzyme. From spin quantitation and computer simulations of the various EPR signals a ratio close to 1:1 for the low-spin/high-spin heme centers in NOR was estimated. Shifting the pH from 8.5 to 5.0, replacing Triton X-100 by other detergents, or adding soybean phospholipids to the protein, led to pronounced changes of the EPR signals in the g = 6 region. In contrast, the strong inhibitor oleic acid did not cause significant spectral changes. NOR which had been reduced by L-ascorbate/phenazine methosulfate prior to incubation with its substrate NO gave the characteristic Fe(II) nitrosyl triplet centered at g ~ 2.01, with a hyperfine splitting of 1.70 mT. In the absence of dioxygen, NOR was quantitatively reduced by either sodium dithionite, or photochemically with deazaflavin and oxalate; the enzyme was reoxidizable by ferricyanide in a fully reversible reaction. Spectroelectrochemical oxidoreductive titrations gave E'o(versus standard hydrogen electrode) = +322 mV for the cytochrome b and +280 mV for the cytochrome ccomponent.