Biological nitrous oxide formation via iron-nitrosyl intermediates

Nitrous oxide (N2O) participates in ozone layer depletion and has a global warming potential nearly 300 times greater than that of CO2. The atmospheric N2O concentration has surged 120% since the pre-industrial era largely due to increased fertilizer use. An estimated 60–75% of N2O is emitted as a by-product of nitrifying or denitrifying microorganisms that transform ammonia (NH3) to nitrate (NO3–) or NO3– to dinitrogen (N2), respectively. Designing inhibitors for N2O-producing enzymes could mitigate these environmental consequences, but this approach requires knowledge of the enzymatic mechanisms. The data presented will provide evidence for intermediates on the reaction pathways of two N2O-producing metalloenzymes: one enzyme produces N2O by reduction of nitric oxide (NO) while the other proceeds by oxidation of hydroxylamine (NH2OH). In both cases, iron-nitrosyl intermediates directly precede N2O formation.

Flavo-diiron protein (FDP) is a non-heme iron protein that catalyzes the two-electron reduction of 2 molecules of NO to N2O (NOR activity). The active site contains a diiron site adjacent to a flavin mononucleotide (FMN)-binding site. In order to separate the reactions of the diiron site from those of the FMN, the reactivities of a deflavinated and fully-flavinated FDP with nitric oxide were compared. Both enzyme forms produced N2O, indicating that the FMN is not necessary for N–N bond formation. Rapid-mixing techniques combined with vibrational and magnetic spectroscopies were used to characterize iron nitrosyl ({FeNO}n, which is Enemark-Feltham notation; n = number of metal d-electrons plus the unpaired NO electron) intermediates on the FDP NOR pathway. The combined data showed that NO sequentially bound to the two iron sites; a diferrous-mononitrosyl (FeII-{FeNO}7) intermediate preceded formation of a diferrous-dinitrosyl ([{FeNO}7]2) species. Formation of the latter species was required for N2O formation.

Cytochrome (cyt) P460 is a heme-containing enzyme expressed by nitrifying bacteria. This enzyme contains a modified c-type heme active site in which the heme ring is cross-linked with a lysine from the protein. Previous work on cyt P460 identified it as a hydroxylamine (NH2OH) oxidase that produced nitrite (NO2–). However, we found that under anaerobic conditions, the enzyme stoichiometrically oxidizes 2 molecules of NH2OH to N2O. Enzyme kinetics, UV-visible absorption and electron paramagnetic resonance (EPR) spectroscopies were combined to establish a working mechanism of cyt P460 N2O production. In the presence of an oxidant, the cyt P460 ferric-hydroxylamine (FeIII–NH2OH) adduct is oxidized to an {FeNO}6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH2OH, resulting in N2O formation. A new pathway intermediate, a six-coordinate {FeNO}7, was recently found to precede the reactive {FeNO}6 intermediate. This new intermediate provides more insight into the cyt P460 hydroxylamine oxidase mechanism as well as the reactivity of iron-nitrosyls.

Jonathan Caranto, PhD (Cornell University)