Warncke K, Dutton P L
Johnson Research Foundation, University of Pennsylvania, Philadelphia 19104.
Biochemistry. 1993 May 11;32(18):4769-79. doi: 10.1021/bi00069a011.
The native ubiquinone-10 redox cofactor has been removed from the QA site of the isolated reaction center protein from Rhodobacter sphaeroides and reconstitution attempted with 28 non-quinone molecules in order to identify factors governing cofactor function and the selectivity displayed by the site in the electron transfers that it catalyzes. Equilibrium binding, in situ electrochemistry, and the kinetics of electron transfer to and from the QA site occupant were examined. Four classes of non-quinone molecules are distinguished according to their ability to occupy the QA site and conduct intraprotein electron transfers. The minimal requirements for occupancy of the QA site are at least one ring and a heteroatom hydrogen bond acceptor. Thus, binding at the site is not highly selective. The rates of electron transfers to and from the class of non-quinone molecules (four) that satisfy the criteria for cofactor function at the QA site compare well with rates previously determined from 14 to 295 K for 14 quinone replacements with comparable values of the reaction free energy. This indicates that the rates are relatively insensitive to variations in exotic and quinone cofactor reorganization energy and the vibrational frequencies coupled to the electron transfers, and that the exotic and quinone cofactors are bound in the QA site in comparable positions. It appears that any variation in rate is determined predominantly by the value of the reaction free energy. The QA site protein-cofactor solvation contribution to the in situ electrochemical potential is roughly constant for 12 rigid quinone and 2 exotic cofactors (average value-61 +/- 2 kcal/mol). Favorable electrostatic contributions governing the reaction free energy are therefore also relatively insensitive to cofactor structure. However, flexible molecules appear to encounter in situ steric constraints that lower the electron affinity by destabilizing the reduced cofactor species. This is a strong determinant of whether a molecule, once in the QA site, will function. These findings compare well with those from studies of electron transfers in synthetic systems.
天然的泛醌 - 10氧化还原辅因子已从球形红杆菌分离的反应中心蛋白的QA位点去除，并尝试用28种非醌分子进行重构，以确定控制辅因子功能的因素以及该位点在其催化的电子转移中所表现出的选择性。研究了平衡结合、原位电化学以及QA位点占据者的电子转移动力学。根据它们占据QA位点并进行蛋白质内电子转移的能力，区分出四类非醌分子。占据QA位点的最低要求是至少一个环和一个杂原子氢键受体。因此，该位点的结合选择性不高。与之前在14至295K下对14种具有可比反应自由能值的醌替代物所确定的速率相比，满足QA位点辅因子功能标准的那类非醌分子（四种）的电子转移速率相当。这表明速率对奇异和醌辅因子重组能的变化以及与电子转移耦合的振动频率相对不敏感，并且奇异和醌辅因子在QA位点中以可比位置结合。似乎速率的任何变化主要由反应自由能的值决定。对于12种刚性醌和2种奇异辅因子，QA位点蛋白质 - 辅因子溶剂化对原位电化学势的贡献大致恒定（平均值 - 61±2千卡/摩尔）。因此，控制反应自由能的有利静电贡献对辅因子结构也相对不敏感。然而，柔性分子似乎会遇到原位空间位阻，通过使还原的辅因子物种不稳定来降低电子亲和力。这是一个分子一旦进入QA位点是否会发挥作用的重要决定因素。这些发现与合成系统中电子转移研究的结果相当。
