Sato Wataru, Hitaoka Seiji, Inoue Kaoru, Imai Mizue, Saio Tomohide, Uchida Takeshi, Shinzawa-Itoh Kyoko, Yoshikawa Shinya, Yoshizawa Kazunari, Ishimori Koichiro
From the Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628.
the Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 819-0315.
J Biol Chem. 2016 Jul 15;291(29):15320-31. doi: 10.1074/jbc.M115.708065. Epub 2016 May 13.
Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.
基于对电子转移反应稳态动力学的突变效应以及我们对相互作用位点的核磁共振分析（坂本健、神谷真、今井真、新泽伊东、内田智、川野健、吉川幸司和石森健（2011年）《美国国家科学院院刊》108卷，12271 - 12276页），我们确定了在周转条件下细胞色素c（Cyt c）与细胞色素c氧化酶（CcO）之间电子转移复合物的结构，并从能量角度对复合物形成所必需的相互作用进行了表征。通过蛋白质对接模拟预测的复合物结构经计算筛选，并通过电子转移反应中突变型Cyt c与CcO的实验动力学数据进行验证。使用选定的Cyt c - CcO复合物结构进行的相互作用分析揭示了每个氨基酸残基对复合物形成所需自由能的静电和疏水贡献。几个带电荷的残基表现出很大的不利（去溶剂化）静电相互作用，这些相互作用几乎被很大的有利（库仑）静电相互作用抵消，但导致了复合物的不稳定。剩余的不稳定自由能由疏水氨基酸残基介导的范德华相互作用补偿，从而形成稳定的复合物。因此，疏水相互作用是周转条件下促进Cyt c与CcO之间复合物形成的主要因素，而静电去稳定自由能的变化提供了突变体中结合自由能的差异。相互作用位点中有利和不利静电相互作用的分布决定了Cyt c在CcO上的结合方向。
