ELECTRON-TRANSFER PROCESSES IN IRON AND COPPER PROTEINS

铁和铜蛋白质中的电子转移过程

• 批准号: 3226253
• 负责人: HARRY B GRAY
• 金额: $ 20.61万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3226253
• 关键词: active sites; azurin; chemical bond; chemical kinetics; chemical structure function; conformation; copper; cytochrome b; electron transport; heavy metals; hemoprotein structure; histidine; lasers; ligands; luminescence; metal complex; metalloproteins; molecular site; myoglobin; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; photochemistry; protein engineering; protein structure function; ruthenium; site directed mutagenesis; spectrometry

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.

在本申请中描述的实验被设计为提供一种方法， 清楚地了解调节的化学和物理参数 蛋白质中的电子转移（ET）反应。 ET反应是许多生物过程中的关键步骤。 生物电子转移的特点是几个独特的功能 在小分子反应中没有发现：反应在低温度下进行 驱动力（小于200 mV），但具有高效率;氧化还原 位点往往是埋在多肽基质内的金属中心， 通常具有暴露的金属配体边缘;由于这种肽鞘， 氧化还原中心通常相隔很长距离（10-25埃）; 结合位点的特异性限制了 蛋白质和氧化还原位点。 因此，挑战在于 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，分子内ET的系统研究 将用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。 在这三起案件中， 定点诱变将用于特异性地制备蛋白质 旨在描述核重定向的作用，并揭示 蛋白质长程电子给体-受体耦合机制 ET反应。 一种测定分子内内皮素的新技术 将制定费率。 表面结合的发光金属配合物 金属蛋白的残余物将被激光脉冲激发并被淬灭 不可逆ET试剂。 基于蛋白质的淬灭产物将 通过分子内ET放松。 表面结合的化学修饰 金属络合物将改变分子内ET的驱动力 反应 本文研究了电子转移速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， 反应的电子耦合强度。 两种型号的电子 已经进化：一个预测，耦合强度呈指数衰减 与直接施主-受主距离;和其他建议，电子 偶联是沿着沿着特定途径（包括共价和 非共价相互作用）。 为了解决 本课题将采用定点诱变方法制备突变体 可以通过将金属络合物连接到选择的蛋白质上来修饰的蛋白质， 表面残留物。 具有相似直接供体-受体的修饰蛋白质 分离，但可以是显著不同的耦合途径。 鉴定 天青蛋白的Q12 H和K122 H突变体说明了这一点。 指数衰减模型预测两者的ET率几乎相同 衍生物;途径模型预测Q21 H的速率 如果K122 H衍生物的系数为 10的四次方 这种类型的比较将澄清 在蛋白质中的远程ET反应中的电子耦合机制。