The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4_D251N and CYP199A4_T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4_D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4_T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Graphic abstract

中文翻译：

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结构见解对细胞色素P450酶中双氧激活所需的酸-醇残基对的作用。

细胞色素P450血红素单加氧酶通常在I螺旋内使用一对酸-醇残基来激活与铁结合的双氧。这项工作旨在澄清有关酒精功能在此过程中的重要性的相互矛盾的报道。制备了P450的突变体CYP199A4（CYP199A4 _D251N和CYP199A4 _T252A），表征并解析了其晶体结构。CYP199A4的酸残基不属于盐桥网络的一部分，这是范例模型系统P450cam的关键功能。取而代之的是通过水分子链直接质子传递网络延伸到表面。然而，CYP199A4 _D251N大大降低了该酶的活性，与质子传递中的作用一致。CYP199A4 _T252A降低了酶的偶联效率，同时增加了过氧化氢的解偶联途径。但是，这种突变的影响远不如P450cam报道的那么明显。与P450cam系统相比，其晶体结构在I螺旋处显示的变化更少。在此P450中似乎不需要在氧激活期间在P450cam的I螺旋内观察到的结构变化。这些差异是由于P450cam中不存在第253位的第二个苏氨酸残基。苏氨酸形成氢键网络的一部分，导致细微的结构变化，并且也存在于大多数P450超家族中。总体，图形摘要