Computation of electron transfer properties for heme-containing oxidoreductases

原子计算机模拟是为了提高对电子传递蛋白中长程电子转移(ET)反应的定量理解。这项工作的开展是为了辅助和补充这一领域的实验工作。现有的计算机模拟技术被扩展到计算控制生物ET反应速率的关键参数。这些参数通常是未知的，而且通常很难测量。利用数值方法，我们的目标是提供可用于生物电子载流子电子隧穿模拟的定量估计。此外，利用分子模拟的微观信息，分析了单个氨基酸残基和周围溶剂对ET率的贡献，并用来解释蛋白质突变的影响，以指导高效仿生药物的设计。在短期内，模拟方法在简单的和实验上很好地表征的电子传递蛋白(特定目标I)上得到了验证。在中长期内，研究了更复杂的血红素过氧化氢酶中的氧化还原和ET反应(特定目标II)。研究方案(具体目标一)：只有在供体和受体具有明确结构的极少数生物ET反应中才能获得可靠的实验ET参数。在这些系统中有Ru修饰的细胞色素(Cyt)蛋白，如Cytc、肌红蛋白、Cytb5和Cytb562。这些蛋白质有丰富的结构、热力学和动力学数据，这使它们可能成为评估计算机模拟准确性的最佳基准系统。第一个目标是计算ET的参数，从位于蛋白质内部的血红素基团到位于蛋白质表面的Ru络合物。对复杂程度越来越高的模型进行了蛋白质的模拟。根据与实验数据的偏差，探索了改进模拟方法的新思路。分析了不同细胞色素的有限温度运动，考察了不同蛋白质折叠和不同的血红素基团对ET参数的影响。此后，研究的重点将转移到下面描述的更复杂的血红素过氧化氢酶上。研究方案(特定目标II)：通过催化过氧化氢在氧气和水中的分解，防止细胞受到氧化损伤。在一定条件下，如果反应中间体化合物I发生单电子还原为化合物II，则其催化活性会急剧降低甚至丧失。最近的结晶学和计算研究表明，含有幽门螺杆菌过氧化氢酶(HPC)的氧化态血红素b形成催化活性较低的化合物II，而含有P.vitale过氧化氢酶的血红素d保持在活性化合物I中。当然，问题是这种差异是与不同的血红素组有关，还是与两种过氧化氢酶的不同蛋白质结构有关。使用在简单细胞色素蛋白上验证的方法(如上所述)，我们建议计算HPC和PVC的ET参数，并确定辅因子、蛋白质和溶剂的相应贡献。下一步，识别血红素基团附近的可电离基团，并计算从可电离基团到氧化的血红素中心的ET参数。我们希望在这些计算的基础上，能够解释HPC和PVC形成催化活性较低的化合物II的不同趋势。

Atomistic computer simulations are proposed to advance aquantitative understanding of long-range electron transfer (ET) reactionsin electron transport proteins. This work is carried out to aid and complementexperimental work in this field. Existing computer simulation techniques areextended to compute key parameters that govern the rate of biologicalET reactions. These parameters are often unknown and usually ratherdifficult to measure. Using numerical methods we aim at providingquantitative estimates that can be used in electron tunneling simulations ofbiological electron carriers. Moreover, exploiting the microscopic information ofmolecular simulations, the contributions of single amino acid residues andthe surrounding solvent to the ET rate are analyzed and used to interpret effects ofprotein mutations that could guide the design of efficient biomimetics. In the short term the simulation methods are validated on simple and experimentally well characterized electron transport proteins (specific aim I). In the medium to long term redox and ET reactions in the more complex heme catalases are investigated (specific aim II). Research programme (specific aim I): Reliable experimental ET parameters areavailable for only very few biological ET reactions where donor and acceptor havea well defined structure. Among these systems are ruthenium modified cytochrome (cyt) proteins such as cyt c, myoglobin, cyt b5 and cyt b562. A wealth of structural, thermodynamic and kinetic data available for these proteins, which makes them possibly the best benchmark systems for assessment of the accuracy ofcomputer simulations. First objective is the computation of ET parametersfor ET from the heme group located inside the proteinto the ruthenium complex located at the surface of the protein. The simulationsof the proteins are carried out for models with increasing degree of complexity. Depending on the deviation with experimental data, new ideas for improvement of the simulation methodology are explored. The finite temperature motion of the different cytochromes are analyzed and used to investigate the influence of the different protein folds and heme groups on the ET parameters. Thereafter the focus of research will shift to the more complicated heme catalases described below.Research programme (specific aim II):Heme catalases prevent cells from oxidative damage by catalyzing the decomposition of hydrogen peroxide in oxygen and water. The catalytic activity is drasticallyreduced or even lost under certain conditions, if reaction intermediatecompound I undergoes one-electron reduction to compound II.Recent crystallographic and computational studies have given evidencethat the oxidized form of heme b containing catalase of H. pylori (HPC) formsthe catalytically less active compound II whereas the heme d containing catalase of P. vitale remains in theactive compound I form. Naturally, the question arises whether this difference is related to the different heme groups or to the different protein structure of the two catalases. Using the methods validated on simple cytochrome proteins (see above), we propose to calculate ET parameters for HPC and PVC and to determine the corresponding contributions of cofactor, protein and solvent.In the next step ionizable groups in the vicinity of the heme groups are identified and the ET parameters computed for ET from the ionizable group to the oxidized heme center. We hope that on the basis of these calculations one can explain the different tendencies of HPC and PVC to form the catalytically less active compound II.