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.

中文翻译：

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细胞色素P460中的血红素-赖氨酸交联对氧化还原催化和一氧化氮敏感性的影响†

氨（NH ₃）氧化菌（AOB）导出用于生命总能量从NH的多电子氧化₃为亚硝酸盐（NO ₂ ^- ）。这种代谢的一种专性中间体是羟胺​​（NH ₂ OH），它可以被AOB酶细胞色素（cyt）P460氧化成有效的温室效应一氧化二氮（N ₂ O）。现在，我们已经在细胞色素P460的NH ₂ OH氧化途径上以光谱形式表征了6坐标（6c）{FeNO} ⁷中间体。这个物种有两个命运：它可以被氧化成{FeNO} ⁶，然后受到NH ₂ OH的攻击最终产生N _2。O，否则它可能会失去其轴向His配体，从而生成稳定的，偏离路径的5坐标（5c）{FeNO} ⁷种。我们显示野生型（WT）cyt P460表现出缓慢的一氧化氮（NO）独立转化（k _His-off = 2.90×10 ^-3 s ^-1），而交联缺陷的Lys70Tyr cyt P460突变蛋白接受他离解经由两者NO无关（ķ_他断= 3.8×10 ^-4小号^-1）和NO依赖性途径[ ķ_{他断（NO）} = 790米之^-1小号^-1]。对这两种蛋白质的NO依赖性途径的Eyring分析表明，在交联缺陷型突变体中，活化熵（ΔS ^‡）明显更大（约27 cal mol ^-1 K ^-1）。我们的结果表明，Lys-血红素交联赋予血红素P460辅因子以刚性，以避免快速的NO依赖性His分解途径和随后形成的非通路5c {FeNO} ⁷物种。还讨论了这些发现与NO信号蛋白（如一氧化二氮/氧结合（H-NOX））的相关性。