Upon further analysis, neither cytochrome c (554) from Nitrosomonas europaea nor its F156A variant display NO reductase activity, though both proteins bind nitric oxide reversibly

作者：McGarry Jennifer M; Pacheco A Andrew^*

来源：Journal of Biological Inorganic Chemistry, 2018, 23(6): 861-878.

DOI：10.1007/s00775-018-1582-4

• Multi-heme cytochromes
• Nitric oxide reduction
• Mechanistic studies

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c (554) (C-554) 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 C-554, first order in NO, and second-order overall. The rate constant for NO binding to the heme is 3000 +/- 140 M(-1)s(-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 C-554-catalyzed NO reduction, either with or with Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when 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 C-554 is crowded by three non-bonding hydrophobic amino acids. After replacing one of these (Phe156) with the smaller alanine, the nitrosylation rate for F156A(2-) and F156A(4-) was about 400x faster than for the wild type, though the rate of the reverse denitrosylation process was almost unchanged. Unlike in the wild-type C-554, the 6-coordinate low-spin hemes of F156A(4-) 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.

• 出版日期2018-8