Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.
Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.
复制标题
通过细胞色素c氧化酶中泵送质子的途径缓慢的质子转移会导致酶的自杀性失活。
DOI：
10.1021/bi0475774
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Hosler,JonathanP
中科院分区：
文献类型：
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作者：
Mills,DeniseA;Hosler,JonathanP
在亚基III的情况下，由于在稳态周转期间经历不可逆失活的可能性增加，theaa 3型细胞色素氧化酶表现出缩短的催化寿命（周转总数）。失活是由亚基I中血红素3 − CuB活性位点的结构改变引起的[Hosler（2004）Biochim. Biophys. Acta 1655，332 - 339]。亚基III的不存在也显著减缓了通过D质子途径到活性位点的质子摄取，以及抑制连接活性位点/质子泵与氧化酶复合物的外表面的质子回流/离开途径。在这里，我们证明了这些现象是相互关联的：通过这些途径将质子缓慢传递到活性位点会导致自杀失活，从而缩短酶的催化寿命。抑制D途径而不是K途径的突变增加了自杀失活的可能性。对D途径的强抑制允许即使在亚基III存在下也发生自杀失活。花生四烯酸通过D途径刺激质子摄取，延缓自杀失活。在Δ Δ pH和ΔpH存在的情况下，抑制质子从蛋白质内表面摄取的稳态转换增强了自杀失活。通过影响D途径和质子回流/退出途径的双突变同时抑制蛋白质两侧的质子摄取，即使在亚基III存在下也大大缩短了氧化酶的催化寿命。因此，通过D途径和回流/出口途径维持快速质子转移是亚基III正常发挥作用以防止细胞色素氧化酶自杀失活的一种机制。实验表明，增加寿命的hemea 3 oxoferryl中间体以及阴离子形式的Glu 286的D途径导致自杀失活的活性位点。
In the absence of subunit III theaa3-type cytochromecoxidase exhibits a shortened catalytic life span (total number of turnovers) due to an increased probability of undergoing irreversible inactivation during steady-state turnover. Inactivation results from structural alteration of the hemea3−CuBactive site in subunit I [Hosler (2004)Biochim. Biophys. Acta 1655, 332−339]. The absence of subunit III also dramatically slows proton uptake to the active site via the D proton pathway, as well as inhibiting the proton backflow/exit pathway that connects the active site/proton pump with the outer surface of the oxidase complex. Here we demonstrate that these phenomena are linked: slow proton delivery to the active site through these pathways induces suicide inactivation, thus shortening the catalytic life span of the enzyme. Mutations that inhibit the D pathway, but not the K pathway, increase the probability of suicide inactivation. Strong inhibition of the D pathway allows suicide inactivation to occur even in the presence of subunit III. Arachidonic acid, which stimulates proton uptake by the D pathway, retards suicide inactivation. Steady-state turnover in the presence of ΔΨ and ΔpH, which inhibits proton uptake from the inner surface of the protein, enhances suicide inactivation. Simultaneous inhibition of proton uptake from both sides of the protein by a double mutation affecting the D pathway and the proton backflow/exit pathway greatly shortens the catalytic life span of the oxidase even in the presence of subunit III. Thus, maintenance of rapid proton transfer through the D pathway and the backflow/exit pathway is one mechanism by which subunit III normally functions to prevent suicide inactivation of cytochromecoxidase. The experiments suggest that increased lifetimes of the hemea3oxoferryl intermediates as well as the anionic form of Glu286 of the D pathway cause suicide inactivation in the active site.