Murphy Emma J, Metcalfe Clive L, Basran Jaswir, Moody Peter C E, Raven Emma Lloyd
Department of Chemistry, Henry Wellcome Building, University of Leicester, University Road, Leicester, LE1 9HN, England U.K.
Biochemistry. 2008 Dec 30;47(52):13933-41. doi: 10.1021/bi801480r.
The binding of substrates to heme enzymes has been widely assumed to occur at the so-called delta-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the gamma-heme edge, is also possible. In previous work [Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307], we showed that binding of ascorbate to ascorbate peroxidase occurred at the gamma-heme edge. Here, we show that the closely related cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of cytochrome c peroxidase shows ascorbate bound to the gamma-heme edge, with hydrogen bonds to the heme propionate and Arg184. In parallel mechanistic studies in variants incorporating the W191F mutation, we show that a transient porphyrin pi-cation radical in Compound I of cytochrome c peroxidase, analogous to that observed in ascorbate peroxidase, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The results are discussed in terms of our more general understanding of substrate oxidation across other heme proteins, and the emerging role of the heme propionates at the gamma-heme edge.
底物与血红素酶的结合一直被广泛认为发生在所谓的δ-血红素边缘。然而，最近出现了一些例子，其中底物在另一个位点，即γ-血红素边缘的结合也是可能的。在之前的工作中[夏普等人（2003年）《自然结构生物学》10卷，303 - 307页]，我们表明抗坏血酸与抗坏血酸过氧化物酶的结合发生在γ-血红素边缘。在这里，我们表明密切相关的细胞色素c过氧化物酶可以通过在酪氨酸36和天冬酰胺184处引入相对适度的结构变化来复制抗坏血酸过氧化物酶的底物结合特性。因此，细胞色素c过氧化物酶的Y36A/N184R/W191F三突变体的晶体学数据显示抗坏血酸结合在γ-血红素边缘，与血红素丙酸酯和精氨酸184形成氢键。在对包含W191F突变的变体进行的平行机制研究中，我们表明细胞色素c过氧化物酶化合物I中类似抗坏血酸过氧化物酶中观察到的瞬态卟啉π-阳离子自由基能够氧化抗坏血酸，但在稳态条件下，这种中间体衰减太快，无法维持抗坏血酸的有效周转。根据我们对其他血红素蛋白底物氧化的更普遍理解以及γ-血红素边缘处血红素丙酸酯的新作用来讨论这些结果。
