Franco R, Ma J G, Lu Y, Ferreira G C, Shelnutt J A
Department of Biochemistry and Molecular Biology, College of Medicine, Institute for Biomolecular Science and H. Lee Moffit Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA.
Biochemistry. 2000 Mar 14;39(10):2517-29. doi: 10.1021/bi991346t.
Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe(2+) chelation into protoporphyrin IX. Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. On the basis of changes in the UV-vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is substoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive lines and the vinyl vibrational mode in the resonance Raman spectra. Shifts in the resonance Raman lines of free-base and metalated porphyrins bound to the wild-type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle. However, the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decompositions indicate a homogeneous ruffled deformation for the nickel protoporphyrin bound to the wild-type ferrochelatase, whereas both planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free-base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together, these findings suggest that distortion occurs in murine ferrochelatase for some porphyrins, even without metal binding, which is apparently required for the yeast ferrochelatase.
亚铁螯合酶（EC 4.99.1.1）是血红素生物合成途径的末端酶，催化亚铁离子（Fe(2+)）螯合到原卟啉IX中。利用小鼠亚铁螯合酶野生型和工程变体的共振拉曼光谱和紫外可见吸收光谱，研究了铁插入卟啉的结构机制。重组变体（即H207N和E287Q）是将小鼠亚铁螯合酶中保守氨基酸组氨酸-207和谷氨酸-287分别替换为天冬酰胺和谷氨酰胺后的酶。根据枯草芽孢杆菌亚铁螯合酶的三维结构推断，这两个残基都位于酶的活性位点。基于紫外可见吸收光谱的变化，向野生型亚铁螯合酶和H207N变体中添加游离碱或金属化卟啉会产生1:1的复合物，最有可能是活性位点处的单体蛋白结合物种。相比之下，向E287Q中添加卟啉（游离碱或金属化）是亚化学计量的，因为该变体在分离和纯化过程中在活性位点保留了结合的卟啉。共振拉曼光谱中结构敏感谱线变窄和乙烯基振动模式证实了卟啉结合的特异性。与野生型亚铁螯合酶结合的游离碱和金属化卟啉的共振拉曼谱线位移表明卟啉大环存在非平面变形。然而，在未首先确定变形的具体类型之前，无法确定变形的程度。值得注意的是，H207N和E287Q结合的卟啉的非平面变形程度不同。事实上，共振拉曼光谱分解表明，与野生型亚铁螯合酶结合的镍原卟啉存在均匀的褶皱变形，而与H207N结合的卟啉同时存在平面和褶皱构象。也许更有启发性的是内源性E287Q结合卟啉的异常共振拉曼光谱，其结构敏感谱线相对于溶液中游离碱原卟啉的谱线大幅上移。这可以解释为蛋白质构象异构体之间的平衡，其中一种构象有利于高度扭曲的卟啉大环。综上所述，这些发现表明，即使没有金属结合，小鼠亚铁螯合酶中某些卟啉也会发生变形，而酵母亚铁螯合酶显然需要金属结合。
