Role of Subunit III in Cytochrome Oxidase Function

亚基 III 在细胞色素氧化酶功能中的作用

• 批准号: 7171935
• 负责人: JONATHAN P HOSLER
• 金额: $ 27.91万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171935
• 关键词: Active Sites; Affect; Bacteria; Binding; Biological Models; Catalytic Domain; Copper; Cytochrome Oxidase Subunit III; Development; Electron Transport; Engineering; Enzymes; Goals; Homologous Gene; In Vitro; Individual; Integral Membrane Protein; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Metals; Mitochondria; Modification; Molecular Chaperones; Mutagenesis; Mutation; Oxidases; Pathway interactions; Phospholipase; Phospholipids; Physiological; Play; Process; Protein Subunits; Proteins; Proton Pump; Protons; Pump; Rate; Resolution; Rhodobacter sphaeroides; Role; Site; Site-Directed Mutagenesis; Solutions; Structure; System; Systems Analysis; Transmembrane Domain; Yeasts; base; crosslink; cytochrome c oxidase; design; heme a3; mutant; prevent; protein function; research study; suicide inactivation; tool

DESCRIPTION (provided by applicant): Cytochrome c oxidase is one of the key energy-generating proteins that function to pump protons across the inner membrane of mitochondria. The catalytic core of the enzyme is composed of three integral membrane proteins, subunits I, II and III. Subunit III does not play a direct role in electron transfer, but it does affect the function of the proton transfer pathways and the proton pumping mechanism in subunit I. Subunit III also prevents the oxidase from undergoing rapid suicide inactivation during normal catalytic turnover. High resolution structures of cytochrome oxidase show that subunit III is associated with tightly bound phospholipids. Mutagenesis and phospholipase treatment of cytochrome c oxidase of the bacterium Rhodobacter sphaeroides, which is closely related to the mitochondrial oxidase, indicates that these lipids are necessary for binding subunit III to subunit I. This is explained by the oxidase structures which show that four phospholipids are simultaneously coordinated by conserved residues of both subunit I and subunit III. The first aim of this project is to examine the protein-lipid interactions of cytochrome oxidase through the analysis of site-directed mutants designed to interrupt these interactions. These experiments will yield information about how tightly bound phospholipids function to facilitate the binding of membrane proteins. These studies should also reveal what type of inter-subunit interactions between integral membrane proteins are used to transmit structural information. A second aim is to study the process of long-distance proton transfer in cytochrome oxidase, using site-directed mutagenesis of subunit III and subunit I residues. Key questions here are how the protein surface around the entry site of a proton pathway controls the rate of proton transfer and how sensitive is the proton conductive pathway in the interior of the protein to modification of residues that connect the pathway to another subunit. A third aim is to establish what structural changes occur with suicide inactivation in order to understand the mechanism of the inactivation process. A fourth aim is to characterize a copper chaperone that appears to be involved in the insertion of CuB into heme a3-CuB active site of cytochrome oxidase. The mechanism of this assembly process is likely to differ from the assembly of other copper centers in proteins since the active site of cytochrome oxidase is buried within subunit I and within the transmembrane region of the protein.

描述（由申请人提供）：细胞色素c氧化酶是一种关键的能量产生蛋白，其功能是将质子泵过线粒体的内膜。酶的催化核心由三个完整的膜蛋白组成，即亚基I、II和III。亚基III在电子转移中不起直接作用，但它确实影响亚基i中质子转移途径和质子泵送机制的功能。亚基III还可以防止氧化酶在正常催化转换过程中发生快速自杀失活。细胞色素氧化酶的高分辨结构表明亚基III与紧密结合的磷脂相关。与线粒体氧化酶密切相关的球红杆菌细胞色素c氧化酶的诱变和磷脂酶处理表明，这些脂质是将亚基III与亚基I结合所必需的。这可以用氧化酶结构来解释，氧化酶结构表明，四种磷脂同时由亚基I和亚基III的保守残基协调。该项目的第一个目的是通过分析旨在中断这些相互作用的位点定向突变体来检查细胞色素氧化酶的蛋白质-脂质相互作用。这些实验将提供关于紧密结合的磷脂如何促进膜蛋白结合的信息。这些研究还应该揭示完整膜蛋白之间的亚基间相互作用用于传递结构信息的类型。第二个目的是研究细胞色素氧化酶的远距离质子转移过程，利用亚基III和亚基I残基的定点诱变。这里的关键问题是质子通路进入位点周围的蛋白质表面如何控制质子转移的速率，以及蛋白质内部的质子传导通路对连接该通路与另一个亚基的残基的修饰有多敏感。第三个目标是确定自杀失活时发生的结构变化，以便了解失活过程的机制。第四个目的是表征一种铜伴侣，它似乎参与了将CuB插入到细胞色素氧化酶的血红素a3-CuB活性位点。这种组装过程的机制可能不同于其他铜中心在蛋白质中的组装，因为细胞色素氧化酶的活性位点埋在蛋白质的亚基I和跨膜区域内。

项目成果

The roles of Rhodobacter sphaeroides copper chaperones PCu(A)C and Sco (PrrC) in the assembly of the copper centers of the aa(3)-type and the cbb(3)-type cytochrome c oxidases.

球形红杆菌铜伴侣 PCu(A)C 和 Sco (PrrC) 在 aa(3) 型和 cbb(3) 型细胞色素 c 氧化酶铜中心组装中的作用。

• DOI: 10.1016/j.bbabio.2012.01.003
• 发表时间: 2012
• 期刊: Biochimica et biophysica acta
• 作者: Thompson,AudieK;Gray,Jimmy;Liu,Aimin;Hosler,JonathanP
• 通讯作者: Hosler,JonathanP

Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.

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
• 发表时间: 2005
• 期刊: Biochemistry.
• 作者: Mills,DeniseA;Hosler,JonathanP
• 通讯作者: Hosler,JonathanP

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.

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
• 发表时间: 2006
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Varanasi,Lakshman;Mills,Denise;Murphree,Anna;Gray,Jimmy;Purser,Chris;Baker,Rodney;Hosler,Jonathan
• 通讯作者: Hosler,Jonathan

Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase.

球形红细菌细胞色素 c 氧化酶 D 途径的替代初始质子受体。

• DOI: 10.1021/bi102002v
• 发表时间: 2011
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Varanasi,Lakshman;Hosler,Jonathan
• 通讯作者: Hosler,Jonathan

Disease-related mutations in cytochrome c oxidase studied in yeast and bacterial models.

在酵母和细菌模型中研究的细胞色素 C 氧化酶的疾病相关突变。

• DOI: 10.1046/j.1432-1033.2003.03482.x
• 发表时间: 2003
• 期刊: European journal of biochemistry
• 作者: Bratton,Melyssa;Mills,Denize;Castleden,CKate;Hosler,Jonathan;Meunier,Brigitte
• 通讯作者: Meunier,Brigitte