Ferrari Davide, Merli Angelo, Peracchi Alessio, Di Valentin Marilena, Carbonera Donatella, Rossi Gian Luigi
Department of Biochemistry and Molecular Biology, University of Parma, Parco Area delle Scienze, 23/A, 43100 Parma, Italy.
Biochim Biophys Acta. 2003 Apr 11;1647(1-2):337-42. doi: 10.1016/s1570-9639(03)00092-x.
Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.
（1）反硝化副球菌甲胺脱氢酶（其含有色氨酸-色氨酸醌（TTQ）作为辅因子）与其氧化还原伙伴蓝铜蛋白氨腈蛋白之间的二元复合物；（2）相同的两种蛋白质与细胞色素c-551i之间的三元复合物。连续波电子顺磁共振已被用于比较这两个系统多晶粉末中铜的状态。虽然从还原的TTQ到铜的催化作用和分子间电子转移太快，我们的测量无法检测到，但血红素还原过程持续几分钟。观察到的速率常数比在溶液中低约四个数量级。对这个表观常数的温度依赖性分析提供了与两个中心之间的电子耦合相关的参数H(AB)以及重组能λ的值，这些值与电子转移是速率决定步骤相一致。根据这些参数以及铜和血红素之间的已知距离，可以计算出取决于中间介质性质的参数β，得到一个典型的跨蛋白质基质电子转移的值。这些发现表明，由于蛋白质动力学的作用尚未明确，溶液中的三元复合物可能比刚性晶体结构具有更高的效率。
