In ammonia oxidizing bacteria (AOB), the integral membrane protein ammonia monooxygenase (AMO) hydroxylates ammonia (NH3) to hydroxylamine (NH2OH). This intermediate is then bound by hydroxylamine oxidoreductase (HAO), an octaheme enzyme containing the heme P460 cofactor. HAO catalyzes the oxidation of NH2OH to nitric oxide (NO) through a short-lived {FeNO}6 intermediate. Another enzyme expressed by AOB- although likely not part of the primary metabolism- is cytochrome (cyt) P460, a homodimer containing one heme P460 cofactor per subunit. Similar to HAO, cyt P460 oxidizes NH2OH to an {FeNO}6 intermediate. Rather than releasing NO, the {FeNO}6 intermediate is stable long enough to react with a second equivalent of NH2OH to produce N2O, a greenhouse gas. It was recently shown that the outer coordination sphere of cyt P460 plays an important role in the oxidation of NH2OH, where a distal glutamate (Glu) residue can reorient in the active site to participate in hydrogen transfer. It was also shown crystallographically that a phenylalanine (Phe) capping the active site could rotate in and out upon substrate binding, possibly acting as a gatekeeping residue. In the present study, the role of the capping Phe in substrate/product binding affinities and the overall mechanism of cyt P460 was investigated by mutating it to alanine (Ala). Substitution of the Phe for Ala is expected to result in a more exposed active site, which would allow both the substrate and the product to diffuse more easily. The effects of this mutation on binding affinities for substrate and products, steady-state kinetics, and product selectivity were tested. Parallels are drawn between the reactivity of this cyt P460 variant and the primary metabolic enzyme HAO.

Futility vs fuel: Pushing cyt P460 from nitrous oxide to nitric oxide production

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.