Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force.

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

Does the oxidation of nitric oxide by oxyMyoglobin share an intermediate with the metMyoglobin-catalyzed isomerization of peroxynitrite?

The reaction of nitric oxide with oxy-myoglobin (oxyMb) to form ferric myoglobin (metMb) and nitrate, and the metMb-catalyzed isomerization of peroxynitrite to nitrate, have long been assumed to proceed via the same iron-bound peroxynitrite intermediate (metMb(OONO)). More recent research showed that the metMb-catalyzed isomerization of peroxynitrite to nitrate produces detectable amounts of nitrogen dioxide and ferryl myoglobin (ferrylMb). This suggests a mechanism in which the peroxynitrite binds to the metMb, ferrylMb is transiently generated by dissociation of NO2, and nitrate is formed when the NO2 nitrogen attacks the ferrylMb oxo ligand. The presence of free NO2 and ferrylMb products reveals that small amounts of NO2 escape from myoglobin's interior before recombination can occur. Free NO2 and ferrylMb should also be generated in the reaction of oxyMb with NO, if the common intermediate metMb(OONO) is formed. However, this report presents a series of time-resolved UV/vis spectroscopy experiments in which no ferrylMb was detected when oxyMb and NO reacted. The sensitivity of the methodology is such that as little as 10% of the ferrylMb predicted from the experiments with metMb and peroxynitrite should have been detectable. These results lead to the conclusion that the oxyMb + NO and metMb + ONOO(-) reactions do not proceed via a common intermediate as previously thought. The conclusion has significant implications for researchers that propose a possible role of oxyMb in intracellular NO regulation, because it means that toxic NO2 and ferrylMb are not generated during NO oxidation by this species.