Upon further analysis, neither cytochrome c554 from Nitrosomonas europaea nor its F156A variant display NO reductase activity, though both proteins bind nitric oxide reversibly.

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the two- or four-electron-reduced protein will nitrosylate reversibly. The process is first order in C554, first order in NO, and second-order overall. The rate constant for NO binding to the heme is 3000 ± 140 M-1s-1, while that for dissociation is 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report (Upadhyay AK, et al. J Am Chem Soc 128:4330, 2006), this study found no evidence of C554-catalyzed NO reduction, either with [Formula: see text] or with [Formula: see text] Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when [Formula: see text] was exposed to an excess of NO, but this is believed to arise from partial intramolecular electron transfer that generates {Fe(NO)}8 at heme II. The vacant heme II coordination site of C554 is crowded by three non-bonding hydrophobic amino acids. After replacing one of these (Phe156) with the smaller alanine, the nitrosylation rate for F156A2- and F156A4- was about 400× faster than for the wild type, though the rate of the reverse denitrosylation process was almost unchanged. Unlike in the wild-type C554, the 6-coordinate low-spin hemes of F156A4- oxidized over the course of several minutes after exposure to NO. Concomitant formation of N2O could explain this heme oxidation, though alternative explanations are equally plausible given the available data.