Ferreira Gloria C, Franco Ricardo, Mangravita Arianna, George Graham N
Department of Biochemistry and Molecular Biology, College of Medicine, Institute for Biomolecular Science and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA.
Biochemistry. 2002 Apr 16;41(15):4809-18. doi: 10.1021/bi015814m.
Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into the protoporphyrin IX ring. Ferrochelatases can be arbitrarily divided into two broad categories: those with and those without a [2Fe-2S] center. In this work we have used X-ray absorption spectroscopy to investigate the metal ion binding sites of murine and Saccharomyces cerevisiae (yeast) ferrochelatases, which are representatives of the former and latter categories, respectively. Co(2+) and Zn(2+) complexes of both enzymes were studied, but the Fe(2+) complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co(2+) and Zn(2+) binding to site-directed mutants of the murine enzyme were also studied, in which the highly conserved and potentially metal-coordinating residues H207 and Y220 were substituted by residues that should not coordinate metal (i.e., H207N, H207A, and Y220F). Our experiments indicate four-coordinate zinc with Zn(N/O)(3)(S/Cl)(1) coordination for the yeast and Zn(N/O)(2)(S/Cl)(2) coordination for the wild-type murine enzyme. In contrast to zinc, a six-coordinate site for Co(2+) coordinated with oxygen or nitrogen was present in both the yeast and murine (wild-type and mutated) enzymes, with evidence of two histidine ligands in both. Like Co(2+), Fe(2+) bound to yeast ferrochelatase was coordinated by approximately six oxygen or nitrogen ligands, again with evidence of two histidine ligands. For the murine enzyme, mutation of both H207 and Y220 significantly changed the spectra, indicating a likely role for these residues in metal ion substrate binding. This is in marked disagreement with the conclusions from X-ray crystallographic studies of the human enzyme, and possible reasons for this are discussed.
亚铁螯合酶（EC 4.99.1.1）是血红素生物合成途径的末端酶，催化亚铁离子插入原卟啉IX环。亚铁螯合酶可任意分为两大类：含有[2Fe-2S]中心的和不含[2Fe-2S]中心的。在本研究中，我们使用X射线吸收光谱法研究了小鼠和酿酒酵母（酵母）亚铁螯合酶的金属离子结合位点，它们分别是前一类和后一类的代表。研究了两种酶的Co(2+)和Zn(2+)配合物，但仅对酵母亚铁螯合酶研究了Fe(2+)配合物，因为小鼠酶的[2Fe-2S]中心会干扰分析。还研究了Co(2+)和Zn(2+)与小鼠酶定点突变体的结合，其中高度保守且可能参与金属配位的残基H207和Y220被不应参与金属配位的残基取代（即H207N、H207A和Y220F）。我们的实验表明，酵母中的锌以Zn(N/O)(3)(S/Cl)(1)配位形成四配位，野生型小鼠酶中的锌以Zn(N/O)(2)(S/Cl)(2)配位。与锌不同，酵母和小鼠（野生型和突变型）酶中均存在一个由氧或氮配位的Co(2+)六配位位点，两者均有两个组氨酸配体的证据。与Co(2+)一样，与酵母亚铁螯合酶结合的Fe(2+)也由大约六个氧或氮配体配位，同样有两个组氨酸配体的证据。对于小鼠酶，H207和Y220的突变显著改变了光谱，表明这些残基在金属离子底物结合中可能起作用。这与人类酶的X射线晶体学研究结论明显不同，并讨论了可能的原因。
