HemeCu Oxidase: Calculated Electron and Proton Transfers

描述（由申请人提供）：鉴于蛋白质的三维结构，应该可以定量了解蛋白质的功能特性。所提出的工作将分析在血红素-Cu氧化酶的双核中心（BNC）中从O2还原为水释放的能量如何驱动矢量质子转移，从而产生跨膜电化学梯度。假设是，在BNC键合的反应变化，几何形状和/或电荷分布被传输到蛋白质的其余部分。然后，这改变了质子输入和输出区域中的电离基团和氢键连接性的分布。MCCE（多构象连续静电学）将用于跟踪整个蛋白质的这些变化，提供：（1）反应循环中每个阶段的净蛋白质摄取（可与测量值进行比较）和质子结合的特定位点（难以通过实验确定）。(2)蛋白质环境对反应自由能的改变和对影响化学的残基的鉴定;（3）在每个步骤上产生的跨膜AW和电子转移、质子转移或偶极重排贡献多少的分解;（4）确定可作为质子泵门的构象变化-这将允许质子在反应的不同阶段进入膜的不同侧。有关BNC中明确变化的信息将通过与其他人合作对反应化学进行高水平DFT分析来获得。这些变化将用于驱动目标分子动力学。将用MCCE评估所得结构，以观察BNC处的反应如何修饰蛋白质。计算将确定种间差异，比较牛细胞溴c氧化酶与在P. dinitificans和Rb中发现的细胞溴c氧化酶。sphaero ides和同源的E.大肠杆菌醌醇氧化酶。突变的影响将被预测。蛋白质结构基序，稳定膜蛋白内部的电荷转移和耦合这些电子转移跨膜质子转移将被确定。

DESCRIPTION (provided by applicant): It should be possible to quantitatively understand functional properties of a protein given its 3-dimensional structure. The proposed work will analyze how the energy released from reduction of 02 to water in the binuclear center (BNC) of Heme-Cu oxidases drives vectorial proton transfer, generating a transmembrane electrochemical gradient. The hypothesis is that on reaction changes in BNC bonding, geometry and/or charge distribution are transmitted to the rest of the protein. This then changes the distribution of ionized groups and hydrogen bond connectivity in proton input and exit regions. MCCE (Multi-Conformation Continuum Electrostatics) will be used to follow these changes throughout the protein, providing: (1) net protein uptake at each stage in the reaction cycle (which can be compared with measured values) and specific sites where protons are bound (which is difficult to determine experimentally). (2) modifications of reaction free energies by the protein environment and identification of residues that influence chemistry; (3) transmembrane AW generated on each step and a breakdown of how much is contributed by electron transfer, proton transfer or dipolar rearrangements; (4) identification of conformational changes that could serve as gates in proton pumping - these would permit access of protons to different sides of the membrane in different stages of the reaction. Information about explicit changes in the BNC will be obtained from collaboration with others carrying out high level DFT analysis of the reaction chemistry. These changes will be used to drive targeted molecular dynamics. The resultant structures will be evaluated with MCCE to see how the reaction at the BNC modifies the protein. The calculations will determine interspecies variation comparing the bovine cytocbrome c oxidase to that found in P. dinitificans and Rb. sphaero ides and to the homologous E. coli quinol oxidase. The effects of mutation will be predicted. Protein structural motifs that stabilize charge transfers deep inside membrane proteins and couple these electron transfers to transmembrane proton transfer will be identified.