Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity

Abstract

Ammonia (NH₃)-oxidizing bacteria (AOB) derive total energy for life from the multi-electron oxidation of NH₃ to nitrite (NO₂⁻). One obligate intermediate of this metabolism is hydroxylamine (NH₂OH), which can be oxidized to the potent greenhouse agent nitrous oxide (N₂O) by the AOB enzyme cytochrome (cyt) P460. We have now spectroscopically characterized a 6-coordinate (6c) {FeNO}⁷ intermediate on the NH₂OH oxidation pathway of cyt P460. This species has two fates: it can either be oxidized to the {FeNO}⁶ that then undergoes attack by NH₂OH to ultimately generate N₂O, or it can lose its axial His ligand, thus generating a stable, off-pathway 5-coordinate (5c) {FeNO}⁷ species. We show that the wild type (WT) cyt P460 exhibits a slow nitric oxide (NO)-independent conversion (k_His-off = 2.90 × 10⁻³ s⁻¹), whereas a cross-link-deficient Lys70Tyr cyt P460 mutant protein underwent His dissociation via both a NO-independent (k_His-off = 3.8 × 10⁻⁴ s⁻¹) and a NO-dependent pathway [k_His-off(NO) = 790 M⁻¹ s⁻¹]. Eyring analyses of the NO-independent pathways for these two proteins revealed a significantly larger (ca. 27 cal mol⁻¹ K⁻¹) activation entropy (ΔS^‡) in the cross-link-deficient mutant. Our results suggest that the Lys–heme cross-link confers rigidity to the positioning of the heme P460 cofactor to avoid the fast NO-dependent His dissociation pathway and subsequent formation of the off-pathway 5c {FeNO}⁷ species. The relevance of these findings to NO signaling proteins such as heme-nitric oxide/oxygen binding (H-NOX) is also discussed.