Issue 31, 2023

Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460

Abstract

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 (NH2OH) to nitric oxide (NO). The non-metabolic enzyme cytochrome (cyt) P460 also oxidizes NH2OH, but instead produces nitrous oxide (N2O). While both enzymes use a heme P460 cofactor, they selectively oxidize NH2OH 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 N2O and NO during NH2OH oxidation under aerobic conditions. A picture emerges where the sterics and electrostatics of the side-chain in this capping position control the kinetics of N2O formation and NO binding affinity. This demonstrates how the outer coordination sphere of cyt P460 is tuned not only for selective NH2OH oxidation, but also for the autocatalytic cross-link forming reaction that imbues activity to an otherwise inactive protein.

Graphical abstract: Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460

Supplementary files

Article information

Article type
Edge Article
Submitted
04 may 2023
Accepted
22 iyn 2023
First published
19 iyl 2023
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2023,14, 8295-8304

Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460

M. M. Bollmeyer, S. H. Majer, R. E. Coleman and K. M. Lancaster, Chem. Sci., 2023, 14, 8295 DOI: 10.1039/D3SC02288A

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