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 Enz
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- 作者：Lakshman Varanasi ; Denise Mills ; Anna Murphree ; Jimmy Gray ; Chris Purser ; Rodney Baker ; Jonathan Hosler
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：December 19, 2006
- 年：2006
- 卷：45
- 期：50
- 页码：14896 - 14907
- 全文大小：378K
- 年卷期：v.45,no.50(December 19, 2006)
- ISSN：1520-4995
文摘
Subunit III of the three-subunit catalytic core of cytochrome c oxidase (CcO) contains nometal centers, but it does bind two lipids, within a deep cleft, in binding sites conserved from bacteria tohumans. Subunit III binds to subunit I, where it prevents the spontaneous suicide inactivation of CcO bydecreasing the probability of side reactions at the heme-Cu O2 reduction site in subunit I. Subunit IIIprevents suicide inactivation by (1) maintaining adequate rates of proton delivery to the heme-Cu activesite 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 bindingsites in subunit III disturbs the subunit I-III interface. Then, two lipid binding site mutants were constructedwith 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-F86Adisrupts interactions with the fatty acid tails of both lipids. Once these mutant CcOs are placed into soybeanphospholipid vesicles, where extensive exchange of bacterial for soybean lipids takes place, it is shownthat altering the lipid binding sites mimics a major loss of subunit III, even though subunit III is completelyretained, in that suicide inactivation becomes much more probable. The rate of proton delivery to theactive site remains rapid, ruling out slow proton uptake as the primary reason for increased suicideinactivation upon alteration of the lipid binding sites. We conclude that altering the lipid binding sites ofsubunit III may promote side reactions leading to suicide inactivation by allowing greater movement tooccur in and around the O2 reduction site of subunit I during the catalytic cycle.