Shi Zhen, Franco Ricardo, Haddad Raid, Shelnutt John A, Ferreira Gloria C
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612-4799, USA.
Biochemistry. 2006 Mar 7;45(9):2904-12. doi: 10.1021/bi051907i.
Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode gamma(15), a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.
通过使用一组在假定的卟啉结合环中携带突变的变体，研究了卟啉与小鼠亚铁螯合酶（血红素生物合成途径的末端酶）的结合。利用共振拉曼（RR）光谱，研究了与亚铁螯合酶结合的卟啉的结构特性，特别是关于活性位点环境中发生的卟啉变形。这种变形被认为是将亚铁离子酶促插入卟啉环以形成血红素的关键步骤。我们之前对卟啉与小鼠亚铁螯合酶结合的RR光谱研究使我们提出，即使在没有金属离子底物的情况下野生型酶也会诱导卟啉变形。在这里，我们通过提供证据拓宽了这一观点，即特定的非平面卟啉变形程度有助于亚铁螯合酶及其变体的催化效率。结果还表明，保守的Trp256（小鼠亚铁螯合酶编号）部分负责观察到的卟啉变形。卟啉与亚铁螯合酶变体的结合导致RR面外振动模式γ(15)的强度降低，γ(15)是一种在野生型酶中很强的鞍状模式。特别是，催化效率比野生型酶低1个数量级的变体估计产生的野生型鞍状变形不到30%。这些结果表明，特定的保守环残基（特别是Trp256）直接参与卟啉底物的鞍状变形。
