Altering conserved lipid binding sites in cytochrome c oxidase of Rhodobacter sphaeroides perturbs the interaction between subunits I and III and promotes suicide inactivation of the enzyme.
Altering conserved lipid binding sites in cytochrome c oxidase of Rhodobacter sphaeroides perturbs the interaction between subunits I and III and promotes suicide inactivation of the enzyme.
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改变球形红杆菌细胞色素 c 氧化酶中保守的脂质结合位点会扰乱亚基 I 和 III 之间的相互作用，并促进该酶的自杀失活。
DOI：
10.1021/bi061390q
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Hosler,Jonathan
中科院分区：
文献类型：
--
作者：
Varanasi,Lakshman;Mills,Denise;Murphree,Anna;Gray,Jimmy;Purser,Chris;Baker,Rodney;Hosler,Jonathan
细胞色素氧化酶（CcO）的三亚基催化核心的亚基III不含金属中心，但它结合两个脂质，在一个深裂缝，在结合位点从细菌到人类保守。亚基III与亚基I结合，通过降低亚基I中血红素−Cu O2还原位点发生副反应的可能性来防止CcO的自发自杀失活。亚基III通过（1）保持足够的质子传递速率至血红素-Cu活性位点和（2）在转换过程中稳定活性位点的结构来防止自杀失活[米尔斯和霍斯勒（2005）生物化学44，4656]。在这里，我们首先表明，突变亚基III中保守的脂质结合位点的几个单独的残基会干扰亚基I-III界面。然后，构建了两个脂质结合位点突变体，其在亚基III上具有亲和标签，使得可以分离具有100%亚基III的突变体CcO。R226 A消除了裂缝最外层脂质的磷酸根离子对，而W59 A-F86 A破坏了与两种脂质的脂肪酸尾部的相互作用。一旦这些突变的CCO被放置到大豆磷脂囊泡中，在那里发生广泛的交换细菌的大豆脂质，它表明，改变脂质结合位点模拟亚基III的主要损失，即使亚基III是完全保留的，在自杀失活变得更加可能。质子传递到活性位点的速率仍然很快，排除了缓慢的质子吸收作为脂质结合位点改变后自杀失活增加的主要原因。我们的结论是，改变亚基III的脂质结合位点可能会促进副反应，导致自杀失活，允许更大的运动发生在和周围的O2还原位点的亚基I在催化循环。
Subunit III of the three-subunit catalytic core of cytochromecoxidase (CcO) contains no metal centers, but it does bind two lipids, within a deep cleft, in binding sites conserved from bacteria to humans. Subunit III binds to subunit I, where it prevents the spontaneous suicide inactivation of CcO by decreasing the probability of side reactions at the heme−Cu O2reduction site in subunit I. Subunit III prevents suicide inactivation by (1) maintaining adequate rates of proton delivery to the heme−Cu active site and (2) stabilizing the structure of the active site during turnover [Mills and Hosler (2005)Biochemistry44, 4656]. Here, we first show that mutating several individual residues of the conserved lipid binding sites in subunit III disturbs the subunit I−III interface. Then, two lipid binding site mutants were constructed with an affinity tag on subunit III such that the mutant CcOs could be isolated with 100% subunit III. R226A eliminates an ion pair to the phosphate of the outermost lipid of the cleft, while W59A-F86A disrupts interactions with the fatty acid tails of both lipids. Once these mutant CcOs are placed into soybean phospholipid vesicles, where extensive exchange of bacterial for soybean lipids takes place, it is shown that altering the lipid binding sites mimics a major loss of subunit III, even though subunit III is completely retained, in that suicide inactivation becomes much more probable. The rate of proton delivery to the active site remains rapid, ruling out slow proton uptake as the primary reason for increased suicide inactivation upon alteration of the lipid binding sites. We conclude that altering the lipid binding sites of subunit III may promote side reactions leading to suicide inactivation by allowing greater movement to occur in and around the O2reduction site of subunit I during the catalytic cycle.