Computational Characterization of the Substrate-Binding Mode in Coproporphyrinogen III Oxidase

Oxygen-dependent coproporphyrinogen III oxidase catalyzes the sequential decarboxylation of the propionate substituents present on the A and B rings of coproporphyrinogen III in the heme biosynthetic pathway. Although extensive experimental investigation of this enzyme has already afforded many insights into its reaction mechanism, several key features (such as the substrate binding mode, the characterization of the active site, and the initial substrate protonation state) remain poorly described. The molecular dynamics simulations described in this paper enabled the determination of a very promising substrate binding mode and the extensive characterization of the enzyme active site. The proposed binding mode is fully consistent with the known selectivity of the active site toward substituted tetrapyrroles and explains the lack of activity of the H131A, R135A, D274A, and R275A mutants and the reasons behind the nonoccurrence of catalysis on the C and D rings of the tetrapyrrole. An important role in this binding mode is fulfilled by G276, as its carbonyl oxygen intervenes in the substrate anchoring by hydrogen bonding its ring D pyrrole NH group. The presence of this interaction (which is only possible with the protonated NH pyrrole group) and the absence of positively charged side chains close to the pyrrole nitrogen (which might stabilize the N-deprotonated pyrrole postulated in some mechanistic proposals) show that the pyrrole ring is very unlikely to undergo deprotonation during the catalytic cycle and allow the discrimination between the previously postulated mechanistic proposals.

氧依赖性粪卟啉原III氧化酶（Oxygen-dependent coproporphyrinogen III oxidase）可在血红素生物合成通路中，催化粪卟啉原III的A、B环上的丙酸酯取代基依次发生脱羧反应。尽管针对该酶的大量实验研究已为其反应机制提供了诸多认知，但仍有多项关键特征（如底物结合模式、活性位点表征以及底物初始质子化状态）尚未得到充分阐释。本文所述的分子动力学模拟（molecular dynamics simulations）成功确定了极具潜力的底物结合模式，并对该酶的活性位点完成了全面表征。本次提出的结合模式与已知的活性位点对取代四吡咯（substituted tetrapyrroles）的选择性完全契合，同时解释了H131A、R135A、D274A以及R275A突变体丧失活性的原因，以及四吡咯C、D环无法发生催化反应的内在机制。G276在该结合模式中发挥关键作用：其羰基氧可通过与D环吡咯NH基团形成氢键，参与底物锚定过程。该相互作用仅能在吡咯NH基团质子化的情况下发生，且吡咯氮原子附近不存在可稳定部分机制假说中提出的去质子化吡咯的带正电侧链，上述两点表明吡咯环在催化循环中几乎不会发生去质子化反应，同时可对此前提出的多种机制假说进行甄别。