Mechanism of Dioxygen Reduction by Heme-Copper Oxidases

血红素铜氧化酶还原分子氧的机制

• 批准号: 8059123
• 负责人: OLOF EINARSDOTTIR
• 金额: $ 13.31万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2011-10-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059123
• 关键词: Active Sites; Apoptosis; Biological; Cattle; Cell Respiration; Chemicals; Complex; Cysteine; Dioxygen; Dyes; Electron Transport; Electrons; Escherichia coli; Future; Heart; Heme; Histidine; Hydrogen; Individual; Inflammation; Kinetics; Label; Ligands; Link; Methods; Modeling; Muscle relaxation phase; Mutate; Nitric Oxide; Optics; Oxidants; Oxidases; Pathologic Processes; Pathway interactions; Phenols; Physiological; Play; Process; Protons; Reaction; Regulation; Research; Rhodobacter sphaeroides; Roentgen Rays; Role; Ruthenium; Site; Site-Directed Mutagenesis; Smooth Muscle; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Thermus thermophilus; Time; Tyrosine; Water; Work; absorption; analog; circular magnetic dichroism; copper oxidase; crosslink; cytochrome c oxidase; electronic structure; in vivo; inhibitor/antagonist; insight; mutant; nanosecond; prolylglutamic acid; research study; stop flow technique; thiouredopyrenetrisulfonate; time use; valyltyrosine

DESCRIPTION (provided by applicant): The primary objective of this research is to elucidate the mechanism of electron and proton transfer during the reduction of dioxygen to water by heme-copper oxidases. Our specific aims will focus on 4 problems: 1. The mechanism of the reduction of dioxygen to water by wild-type and mutant bacterial heme-copper oxidases will be studied by the CO flow-flash method. Time-resolved multichannel optical absorption spectroscopy, in conjunction with singular value decomposition (SVD) and global exponential fitting analysis, will be used to follow the kinetics of electron and proton transfer and to deduce the UV-Vis spectra of the transient intermediates. These studies should provide new insight into the mechanism of the dioxygen reduction reaction by heme-copper oxidases. 2. The intramolecular electron transfer in the bacterial oxidases, bo3 from E. coli, aa3 from Rhodobacter sphaeroides and ba3 from Thermus thermophilus, will be investigated using a photoactivatable dye, thiouredopyrenetrisulfonate (TUPS), covalently linked to single reactive cysteine residues on the oxidases. Time-resolved optical absorption spectroscopy will be used to determine the spectra of the intermediates. By varying the distance between the labeled cysteine and the initial electron acceptor and by introducing breaks into presumed electron transfer pathways by site-directed mutagenesis, detailed information regarding intramolecular electron transfer pathways in heme-copper oxidases will be obtained. 3. We will synthesize chemical analogs of the active site of cytochrome oxidase, including the cyclic pentapeptide (His-Pro-Glu-Val-Tyr) with and without Cu-ligands incorporated. The analogs will be studied using steady-state and time-resolved UV-Vis spectroscopy, FTIR and EPR, which will provide insight into the role of the cross-link in cytochrome oxidase function. 4. Nitric oxide (NO) has emerged as an important biological regulatory agent. A new direction in our research is to understand how NO interacts with heme-copper oxidases. The photodissociation dynamics of ruthenium nitrosyl complexes and the reaction of the photoproduced NO with heme-copper oxidases and their turnover intermediates will be investigated using time-resolved multichannel optical absorption spectroscopy. These studies will circumvent rate limitation imposed by stopped-flow techniques and provide information regarding NO regulation of cytochrome oxidase activity.

描述（由申请人提供）：本研究的主要目的是阐明血红素铜氧化酶将分子氧还原为水期间电子和质子转移的机制。我们的具体目标将集中在四个问题上： 1.通过CO流动闪蒸法研究野生型和突变型细菌血红素铜氧化酶将双氧还原成水的机制。时间分辨多通道光学吸收光谱与奇异值分解（SVD）和全局指数拟合分析相结合，将用于跟踪电子和质子转移的动力学，并推导出瞬态中间体的紫外-可见光谱。这些研究应该为血红素铜氧化酶的双氧还原反应机制提供新的见解。 2. 将使用光活化染料硫脲芘三磺酸盐 (TUPS) 来研究细菌氧化酶（大肠杆菌的 bo3、球形红杆菌的 aa3 和嗜热栖热菌的 ba3）中的分子内电子转移，该染料与氧化酶上的单个反应性半胱氨酸残基共价连接。时间分辨光学吸收光谱将用于确定中间体的光谱。通过改变标记的半胱氨酸和初始电子受体之间的距离，并通过定点诱变将断裂引入到假定的电子转移途径中，将获得有关血红素铜氧化酶中分子内电子转移途径的详细信息。 3. 我们将合成细胞色素氧化酶活性位点的化学类似物，包括掺入或不掺入铜配体的环状五肽（His-Pro-Glu-Val-Tyr）。将使用稳态和时间分辨紫外-可见光谱、FTIR 和 EPR 对类似物进行研究，这将深入了解交联在细胞色素氧化酶功能中的作用。 4. 一氧化氮（NO）已成为一种重要的生物调节剂。我们研究的一个新方向是了解 NO 如何与血红素铜氧化酶相互作用。将使用时间分辨多通道光学吸收光谱研究钌亚硝酰配合物的光解动力学以及光产生的 NO 与血红素铜氧化酶及其转换中间体的反应。这些研究将规避停流技术施加的速率限制，并提供有关细胞色素氧化酶活性的 NO 调节的信息。

项目成果

Near-infrared time-resolved optical absorption studies of the reaction of fully reduced cytochrome c oxidase with dioxygen.

• DOI: 10.1021/bi002220v
• 发表时间: 2001-01
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: I. Szundi;Guang-Ling Liao;Ó. Einarsdóttir

Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection.

使用硫脲芘三磺酸盐标记的细胞色素 c 在细胞色素 c/细胞色素 c 氧化酶复合物中进行光诱导电子转移。

• DOI: 10.1021/bi002341v
• 发表时间: 2001
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Szundi,I;Cappuccio,JA;Borovok,N;Kotlyar,AB;Einarsdóttir,O
• 通讯作者: Einarsdóttir,O

Intermediates in the reaction of fully reduced cytochrome c oxidase with dioxygen.

完全还原的细胞色素 C 氧化酶与双氧反应的中间体。

• DOI: 10.1021/bi981092w
• 发表时间: 1998
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Sucheta,A;Szundi,I;Einarsdóttir,O
• 通讯作者: Einarsdóttir,O

Electron transfer rates and equilibrium within cytochrome c oxidase.

细胞色素 c 氧化酶内的电子转移速率和平衡。

• DOI: 10.1046/j.1432-1327.2000.01072.x
• 发表时间: 2000
• 期刊: European journal of biochemistry
• 作者: Farver,O;Einarsdóttir,O;Pecht,I
• 通讯作者: Pecht,I

Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments.

• DOI: 10.1083/jcb.200804140
• 发表时间: 2008-10-06
• 期刊: JOURNAL OF CELL BIOLOGY
• 影响因子: 7.8
• 作者: Caswell, Patrick T.;Chan, May;Lindsay, Andrew J.;McCaffrey, Mary W.;Boettiger, David;Norman, Jim C.
• 通讯作者: Norman, Jim C.