The proton donor for O-O bond scission by cytochrome c oxidase
The proton donor for O-O bond scission by cytochrome c oxidase
复制标题
DOI：
10.1073/pnas.0802512105
复制
发表时间：
2008-08-05
影响因子：
11.1
通讯作者：
Verkhovsky, Michael I.
中科院分区：
文献类型：
--
作者：
Gorbikova, Elena A.;Belevich, Ilya;Verkhovsky, Michael I.
细胞色素c氧化酶是线粒体和许多需氧细菌中耗氧的主要催化剂。氧还原的关键步骤是O - O键的断裂以及由血红素a(3)和Cu - B组成的双核活性位点的中间产物PR的形成。该反应所需质子的供体被认为是一个独特的酪氨酸残基（Tyr - 280），它与Cu - B的一个组氨酸配体共价交联。为了验证这一想法，我们使用了来自反硝化副球菌的Glu - 278 - Gln突变酶，其中与氧的反应在PR中间产物处停止。使用了三种不同的时间分辨技术。光谱学显示PR物质快速（约60微秒）出现，同时血红素a完全氧化，傅里叶变换红外光谱显示在1308 cm⁻¹处有一个谱带，这是交联的Tyr - 280去质子化形式的特征。PR物质形成过程中电势的变化表明质子在垂直于膜平面约4埃的距离上转移，这接近Tyr - 280羟基的氧原子与结合氧之间的距离。这些结果有力地支持了交联的酪氨酸是细胞色素c氧化酶断裂O - O键的质子供体这一假设，并强化了这样一种观点，即在血红素a被氧化的条件下，该酪氨酸也为O₂还原提供第四个电子。
Cytochrome c oxidase is the main catalyst of oxygen consumption in mitochondria and many aerobic bacteria. The key step in oxygen reduction is scission of the O-O bond and formation of an intermediate PR of the binuclear active site composed of heme a(3) and Cu-B. The donor of the proton required for this reaction has been suggested to be a unique tyrosine residue (Tyr-280) covalently cross-linked to one of the histidine ligands of Cu-B. To test this idea we used the Glu-278-Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the PR intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (approximate to 60 mu s) appearance of the PR species along with full oxidation of heme a, and FTIR spectroscopy revealed a band at 1,308 cm(-1), which is characteristic for the deprotonated form of the cross-linked Tyr-280. The development of electric potential during formation of the PR species suggests transfer of a proton over a distance of approximate to 4 angstrom perpendicular to the membrane plane, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr-280 and the bound oxygen. These results strongly support the hypothesis that the cross-linked tyrosine is the proton donor for O-O bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O-2 reduction in conditions where heme a is oxidized.