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Issue 13, 2019
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Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460

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Abstract

Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NH2OH) as a source of reducing equivalents for cellular respiration. Despite disproportionation or violent decomposition being typical outcomes of reactions of NH2OH with iron, AOB and anammox heme P460 proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled, stepwise oxidation of NH2OH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant from the AOB Nitrosomonas sp. AL212 is able to form all intermediates of cyt P460 catalysis, but is nevertheless incompetent for NH2OH oxidation. We now show via site-directed mutagenesis, activity assays, spectroscopy, and structural biology that this lack of activity is attributable to the absence of a critical basic glutamate residue in the distal pocket above the heme P460 cofactor. This substitution is the only distinguishing characteristic of a protein that is otherwise effectively structurally and spectroscopically identical to an active variant. This highlights and reinforces a fundamental principal of metalloenzymology: metallocofactor inner-sphere geometric and electronic structures are in many cases insufficient for imbuing reactivity; a precisely defined outer coordination sphere contributed by the polypeptide matrix can be the key differentiator between a metalloenzyme and an unreactive metalloprotein.

Graphical abstract: Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460

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Supplementary files

Article information


Submitted
13 Jan 2019
Accepted
14 Feb 2019
First published
06 Mar 2019

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2019,10, 3756-3764
Article type
Edge Article

Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460

M. A. Smith, S. H. Majer, A. C. Vilbert and Kyle M. Lancaster, Chem. Sci., 2019, 10, 3756
DOI: 10.1039/C9SC00195F

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