Engineering the Substrate Specificity and Reactivity of a Heme Protein: Creation of an Ascorbate Binding Site in Cytochrome c Peroxidase
Engineering the Substrate Specificity and Reactivity of a Heme Protein: Creation of an Ascorbate Binding Site in Cytochrome c Peroxidase
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DOI：
10.1021/bi801480r
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发表时间：
2008-12-30
期刊：
影响因子：
2.9
通讯作者：
Raven, Emma Lloyd
中科院分区：
文献类型：
--
作者：
Murphy, Emma J.;Metcalfe, Clive L.;Raven, Emma Lloyd
底物与血红素酶的结合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，其中底物结合在另一个位点，即γ -血红素边缘，也是可能的。在以前的工作中[Sharp et al.(2003)]。我们发现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，我们发现密切相关的细胞色素c过氧化物酶可以通过在Tyr36和Asn 184上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三重变体的晶体学数据显示，抗坏血酸与γ -血红素边缘结合，并与丙酸血红素和Arg 184形成氢键。在包含w191f突变的变异的平行机制研究中，我们发现在细胞色素c过氧化物酶的化合物I中有一个短暂的卟啉阳离子自由基，类似于在抗坏血酸过氧化物酶中观察到的，能够氧化抗坏血酸，但在稳定状态下，这种中间产物衰变太快，无法维持抗坏血酸的有效周转。这些结果是根据我们对其他血红素蛋白的底物氧化的更一般的理解，以及血红素丙酸盐在-血红素边缘的新作用来讨论的。
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 Asn 184. 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 Arg 184. In parallel mechanistic studies in variants incorporating the W 191 F 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.