Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, United States.
Biochemistry. 2011 Mar 8;50(9):1535-44. doi: 10.1021/bi101170p. Epub 2011 Feb 10.
The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.
血红素生物合成途径以亚铁螯合酶催化的亚铁离子螯合到原卟啉 IX 中形成原血红素为终点。亚铁螯合酶的催化机制已被提出涉及稳定非平面卟啉以将吡咯氮呈现给金属离子底物。以前，我们假设亚铁螯合酶诱导的卟啉底物的非平面扭曲对纳入卟啉环的二价金属离子具有选择性，并通过降低酶对金属化卟啉的亲和力来促进其释放。现在，使用共振拉曼光谱，检查了与酶的催化性质相关的直接进化的 Ni(2+)-螯合酶变体的活性位点结合的卟啉的结构特性，涉及卟啉变形的模式和程度。直接进化以表现出比亲本野生型亚铁螯合酶更高的 Ni(2+)-螯合酶活性的 Ni(2+)-螯合酶变体（S143T、F323L 和 S143T/F323L）诱导卟啉底物的较弱鞍变形。与野生型亚铁螯合酶相比，进化变体的 Ni(2+)-和 Fe(2+)-螯合酶活性的稳态动力学参数增加。特别是，减少的卟啉鞍变形与对金属离子底物（Ni(2+)或 Fe(2+)）的催化效率增加相关。结果使我们提出，诱导的原卟啉 IX 鞍变形模式的降低与亚铁螯合酶对金属离子的偏好降低和卟啉螯合步骤变慢有关。
