ELECTRON TRANSFER PROCESSES IN IRON AND COPPER PROTEINS

本应用程序中描述的实验旨在提供 对化学和物理参数有清晰的理解 蛋白质中的电子转移反应。 ET反应是许多生物过程中的关键步骤。 生物电子转移有几个独特的特征。 在小分子反应中没有发现：反应在低浓度下进行 驱动力(低于200 mV)，但效率高；氧化还原 然而，位点往往是埋藏在多肽基质中的金属中心 通常带有裸露的金属配体边缘；由于这种肽鞘， 氧化还原中心一般相隔很长的距离(10-25埃)； 结合位点的特殊性制约了分子间的相互定向 蛋白质，以及氧化还原位点。那么，挑战就是如何 了解生物分子的物理和化学性质如何产生 高效的电子传输系统。 在接下来的五年里，对分子内ET的系统研究 将与三种蛋白质进行反应：铜绿假单胞菌 天青蛋白、人肌红蛋白和牛细胞色素b5。在所有三个案例中， 定点突变将被用于制备特定的蛋白质 旨在描述核重新定向的作用并揭示 长程蛋白质中供体-受体电子偶联的机制 ET反应。一种测量分子内ET的新技术 将制定费率。结合在表面的发光金属络合物 金属蛋白的残留物将被激光脉冲激发和猝灭 不可逆的ET试剂。基于蛋白质的猝灭产品将 通过分子内ET放松。表面键合分子的化学修饰 金属络合物会改变分子内ET的驱动力 反应。Et率的驱动力和温度依赖关系 这些修饰的蛋白质将揭示核重组能量和 反应的电子耦合强度。电子产品的两种模式 已经进化：有人预测耦合强度呈指数衰减 与供体-受体的直接距离有关；另一些人则认为电子 偶联是沿着特定的途径(包括共价和 供体和受体之间的非共价作用)。为了解决 本期，将采用定点突变的方法制备突变体 可以通过将金属络合物附着在选定的蛋白质上而进行修饰的蛋白质 表面残留物。具有相似直接供体-受体的修饰蛋白 分离，但显著不同的耦合路径可以是 已确认身份。天青的Q12H和K122H突变体说明了这一点。 指数衰减模型预测两者的外星率几乎相同 导数；路径模型预测Q21H的速度 导数会比K122H导数慢一个系数 10的四次方。这种类型的比较将澄清 蛋白质长程电子转移反应中的电子耦合机制。

The experiments described in this application are designed to provide a clear understanding of the chemical and physical parameters that regulate electron-transfer (ET) reactions in proteins. ET reactions are known to key steps in many biological processes. Biological electron transfers are characterized by several unique features not found in small-molecule reactions: the reactions proceed at low driving forces (less than 200 mV), but with high efficiencies; the redox sites tend to be metal centers buried inside polypeptide matrices, though often with exposed metal-ligand edges; because of this peptide sheath, the redox centers are generally separated by long distances (10-25 angstroms); and the specificity of binding sites restricts the mutual orientations of the proteins, as well as the redox sites. The challenge, then, is to comprehend how the physical and chemical properties of biomolecules produce highly efficient electron-transport systems. During the next five years, systematic investigations of intramolecular ET reactions will be performed with three proteins: Pseudomonas aeruginosa azurin, human myoglobin, and bovine cytochrome b5. In all three cases, site directed mutagenesis will be used to prepare proteins specifically designed to characterize the role of nuclear reorientation and to reveal the mechanism of donor-acceptor electronic coupling in long range protein ET reactions. A new technique for the measurement of intramolecular ET rates will be developed. Luminescent metal complexes bound to surface residues of metalloproteins will be excited with laser pulses and quenched with irreversible ET reagents. The protein-based quenching product will relax via intramolecular ET. Chemical modifications of the surface-bound metal complexes will vary the driving force for the intramolecular ET reaction. The driving-force and temperature dependences of ET rates in these modified proteins will reveal nuclear reorganization energies and electronic coupling strengths for the reactions. Two models for electronic have evolved: one predicts that the coupling strength decays exponentially with direct donor-acceptor distance; and other suggests that electronic coupling is mediated along specific pathways (comprised of covalent and noncovalent interactions) between donor and acceptor. In order to resolve this issue, site directed mutagenesis will be used to prepare mutant proteins that can be modified by attachment of metal complexes to selected surface residues. Modified proteins with similar direct donor-acceptor separations, but significantly different coupling pathways can be identified. The Q12H and K122H mutants of azurin illustrate the point. The exponential-decay model predicts nearly identical ET rates for the two derivatives; the pathway model predicts that the rate for the Q21H derivative will be slower then that if the K122H derivative by a factor of 10 to the fourth power. Comparisons of this type will clarify the mechanism of electronic coupling in long-range ET reactions in proteins.