Fülöp V, Phizackerley R P, Soltis S M, Clifton I J, Wakatsuki S, Erman J, Hajdu J, Edwards S L
Oxford Centre for Molecular Sciences, UK.
Structure. 1994 Mar 15;2(3):201-8. doi: 10.1016/s0969-2126(00)00021-6.
Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group.
Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures.
The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.
酵母细胞色素c过氧化物酶是一种可溶性含血红素蛋白，存在于线粒体电子传递链中，可能在其中起到抵御有毒过氧化物的作用。本研究的目的是获得酵母细胞色素c过氧化物酶与过氧化氢反应中双氧化瞬态中间体（称为化合物I）的可靠结构。该中间体在色氨酸191上含有一个半稳定自由基和一个氧合铁血红素基团。
通过在流通池中使结晶酶与过氧化氢反应，在酵母细胞色素c过氧化物酶晶体中产生了化合物I。在单独的实验中，通过显微分光光度法和劳厄晶体学对反应进行监测。添加过氧化氢后两分钟内几乎完全转化为化合物I，中间体浓度在接下来的半小时内保持在相似水平。通过劳厄衍射确定了中间体的结构。化合物I的精修劳厄结构显示，过氧化物结合位点有明显的结构变化，但自由基位点没有显著变化。使用新的软件包（LEAP）处理照片，克服了以前从劳厄曝光中提取结构信息时遇到的许多问题。
该蛋白质中血红素环境的几何形状使得结构变化极小，其幅度与细胞色素c中Fe2+/Fe3+转变所观察到的相似。结果表明，这些分子在进化过程中能够以最小的结构调整需求来转移电子。
