Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460.
Melissa M BollmeyerSean H MajerRachael E ColemanKyle M LancasterPublished in: Chemical science (2023)
Product selectivity of ammonia oxidation by ammonia-oxidizing bacteria (AOB) is tightly controlled by metalloenzymes. Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine (NH 2 OH) to nitric oxide (NO). The non-metabolic enzyme cytochrome (cyt) P460 also oxidizes NH 2 OH, but instead produces nitrous oxide (N 2 O). While both enzymes use a heme P460 cofactor, they selectively oxidize NH 2 OH to different products. Previously reported structures of Nitrosomonas sp. AL212 cyt P460 show that a capping phenylalanine residue rotates upon ligand binding, suggesting that this Phe may influence substrate and/or product binding. Here, we show via substitutions of the capping Phe in Nitrosomonas europaea cyt P460 that the bulky phenyl side-chain promotes the heme-lysine cross-link forming reaction operative in maturing the cofactor. Additionally, the Phe side-chain plays an important role in modulating product selectivity between N 2 O and NO during NH 2 OH oxidation under aerobic conditions. A picture emerges where the sterics and electrostatics of the side-chain in this capping position control the kinetics of N 2 O formation and NO binding affinity. This demonstrates how the outer coordination sphere of cyt P460 is tuned not only for selective NH 2 OH oxidation, but also for the autocatalytic cross-link forming reaction that imbues activity to an otherwise inactive protein.