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

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.

Fig. 1. Heme P460 cofactors found in N. europaea (a) hydroxylamine oxidoreductase (PDBID: 4FAS) and (b) cytochrome P460 (PDBID: ; 2JE3). Adapted from ref. 22.

Fig. 2. Working mechanism for NH₂OH oxidation by heme P460 cofactors. The steps indicated in black distinguish cyt P460, in which the {FeNO} persists and can undergo nucleophilic attack by NH₂OH. In HAO, no {FeNO} has been observed, presumably due to facile release of NO, its stoichiometric product of NH₂OH oxidation. Adapted from ref. 23.

Fig. 3. Cyt P460 from N. europaea (a, PDB ID: ; 2JE3) and Nitrosomonas sp. AL212 (b, PDB ID: ; 6AMG). The presence of a distal carboxylate (Glu97) in the N. europaea variant marks a key difference from the NH₂OH oxidation-incompetent AL212 variant, which has an alanine (Ala131) occupying the position.

Fig. 4. UV-vis absorption traces showing reactions of 12 μM WT N. sp. AL212 cyt P460 (a) or AL212 Ala131Glu (b) with 10 mM NH₂OH and 70 μM DCPIP. Reactions were carried out at 25 °C in 50 mM sodium phosphate (pH 8.0). Reactions were initiated after collection of red initial traces by addition of NH₂OH, with subsequent gray scans collected at 15 s intervals. (c) Represents a kinetic trace of the absorbance of DCPIP at 605 nm for both WT AL212 and Ala131Glu under the conditions in (a) and (b), with scans every 0.5 seconds.

Fig. 5. Steady-state NH₂OH oxidation activity plot for all investigated cyt P460 variants. Assay conditions were 1 μM cyt P460, 6 μM phenazine methosulfate (PMS), and 70 μM DCPIP. NH₂OH concentrations ranged from 0–20 mM. Assays were carried out anaerobically in 50 mM sodium phosphate, pH 8.0, at 25 °C. Each data point is the average of at least three trials, with error bars representing one standard deviation. The data series in black represents NH₂OH-dependent rates of DCPIP consumption under enzyme-free but otherwise identical conditions.

Fig. 6. Plot of N₂O production as a function of NH₂OH concentration by AL212 cyt P460 variants. Assay conditions were 5 μM cyt P460, 1 mM DCPIP, and NH₂OH concentrations ranging from 0–1 mM. Assays were carried out anaerobically in 200 mM HEPES, pH 8.0. Each data point is the average of at least three trials, with error bars representing one standard deviation.

Fig. 7. EPR spectra of cyt P460 variants. (a) 10 K X-band (9.40 GHz) EPR spectrum (red) and simulated spectra (component 1 in black and component 2 in grey) of Fe^III WT N. sp. AL212 cyt P460, Fe^III Ala131Glu, Fe^III Ala131Gln recorded at 633 μW microwave power. (b) EPR spectrum of NH₂OH-bound Ala131Glu, Ala131Gln, and N. europaea cyt P460 under the same conditions. *Indicates the presence of contamination from the {FeNO} intermediate in both Ala131Glu and N. europaea cyt P460.

Fig. 8. Cyt P460 active site views showing orientation of residue 131. (a) Comparison of 1.97 Å structure of Ala131Glu (white, PBDID: 6EOX) with 2.30 Å structure of Ala131Gln (green, PDBID: ; 6EOZ) active sites with Phe76 and residue 131 highlighted in each. (b) 2.25 Å structure of Ala131Gln with NH₂OH bound (PDB ID: ; 6EOY, chain A). (c) 1.97 Å structure of Ala131Glu with NO bound (PDB ID: ; 6E17 10¹⁷, chain A).

Scheme 1. Proposed N. sp. AL212 Ala131Glu cyt P460 molecular motions accompanying substrate binding.

Fig. 9. Sequence alignment generated using MEGA X software showing homology between cyt P460 genes from N. europaea, the two cyt P460 sequences from N. sp. AL212, and genes predicted from mammalian pathogenic bacteria Burkholderia cepacia, Pseudomonas aeruginosa, and Vibrio metoecus. * indicates conserved Lys cross-link, ** indicates the critical second-sphere Ala/Glu/Phe residue.