Bonagura C A, Sundaramoorthy M, Pappa H S, Patterson W R, Poulos T L
Department of Molecular Biology, University of California, Irvine 92717-3900, USA.
Biochemistry. 1996 May 14;35(19):6107-15. doi: 10.1021/bi960122x.
The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.
细胞色素c过氧化物酶和抗坏血酸过氧化物酶的晶体结构非常相似，包括活性位点结构。两种过氧化物酶都有一个色氨酸残基，称为近端色氨酸，直接位于近端组氨酸血红素配体相邻位置。在催化循环过程中，细胞色素c过氧化物酶中的近端色氨酸被氧化成阳离子自由基。然而，在抗坏血酸过氧化物酶中，被氧化的是卟啉，而不是近端色氨酸，尽管这两种过氧化物酶的活性位点结构非常相似。在抗坏血酸过氧化物酶中，有一个阳离子位于距近端色氨酸约8埃处，但在细胞色素c过氧化物酶中不存在，这被认为是抗坏血酸过氧化物酶不形成色氨酸自由基的一个原因。定点诱变已被用于将抗坏血酸过氧化物酶的阳离子结合位点引入细胞色素c过氧化物酶。晶体结构表明，突变体现在结合了一个阳离子。电子顺磁共振波谱表明，含阳离子的细胞色素c过氧化物酶突变体不再形成稳定的色氨酸自由基。以亚铁细胞色素c为底物时，阳离子突变体的活性不到野生型水平的1%，而对小分子底物愈创木酚的活性则增加。这些结果表明，长程静电效应可以控制氧化还原活性氨基酸侧链的反应性，并且近端色氨酸的氧化/还原在亚铁细胞色素c的氧化中很重要。
