Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Dipartimento di Chimica "Ugo Schiff"-DICUS, Università di Firenze, Sesto Fiorentino, Italy.
Protein Sci. 2023 Nov;32(11):e4788. doi: 10.1002/pro.4788.
Understanding the reaction mechanism of enzymes at the molecular level is generally a difficult task, since many parameters affect the turnover. Often, due to high reactivity and formation of transient species or intermediates, detailed information on enzymatic catalysis is obtained by means of model substrates. Whenever possible, it is essential to confirm a reaction mechanism based on substrate analogues or model systems by using the physiological substrates. Here we disclose the ferrous iron incorporation mechanism, in solution, and in crystallo, by the coproporphyrin III-coproporphyrin ferrochelatase complex from the firmicute, pathogen, and antibiotic resistant, Listeria monocytogenes. Coproporphyrin ferrochelatase plays an important physiological role as the metalation represents the penultimate reaction step in the prokaryotic coproporphyrin-dependent heme biosynthetic pathway, yielding coproheme (ferric coproporphyrin III). By following the metal titration with resonance Raman spectroscopy and x-ray crystallography, we prove that upon metalation the saddling distortion becomes predominant both in the crystal and in solution. This is a consequence of the readjustment of hydrogen bond interactions of the propionates with the protein scaffold during the enzymatic catalysis. Once the propionates have established the interactions typical of the coproheme complex, the distortion slowly decreases, to reach the almost planar final product.
理解酶在分子水平上的反应机制通常是一项艰巨的任务，因为许多参数会影响酶的周转率。通常，由于高反应性和瞬态物种或中间产物的形成，关于酶催化的详细信息是通过模型底物获得的。只要有可能，就必须通过使用生理底物来确认基于底物类似物或模型系统的反应机制。在这里，我们揭示了梭菌病原体和抗生素抗性李斯特菌中粪卟啉原 III-粪卟啉原亚铁螯合酶复合物在溶液中和结晶中的亚铁离子掺入机制。粪卟啉原亚铁螯合酶在金属化过程中起着重要的生理作用，因为金属化代表了原核粪卟啉依赖性血红素生物合成途径中的倒数第二步反应，生成粪卟啉（三价铁粪卟啉 III）。通过共振拉曼光谱和 X 射线晶体学跟踪金属滴定，我们证明在金属化过程中，在晶体中和溶液中，鞍形扭曲都变得占主导地位。这是由于在酶催化过程中，丙酸盐与蛋白质支架的氢键相互作用的重新调整。一旦丙酸盐建立了与粪卟啉复合物典型的相互作用，扭曲就会缓慢减小，达到几乎平面的最终产物。
