Abstract
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.
Highlights
15320 JOURNAL OF BIOLOGICAL CHEMISTRY trostatic interactions in the interaction site determines the orientation of the binding of Cyt c on cytochrome c oxidase (CcO)
A series of electron transfer (ET) reactions is terminated at cytochrome c oxidase (CcO), where molecular oxygen is reduced to water
Site-directed Mutagenesis Experiments on Cyt c—To determine hot spot residues in Cyt c in the interaction with CcO, we performed site-directed mutagenesis on 11 residues (Ile-9, Ile11, Ile-81, Lys-7, Lys-8, Lys-13, Lys-39, Lys-72, Lys-79, Lys-86, and Lys-87) that were predicted to be located at the binding interface of the Cyt c-CcO complex [13]
Summary
Protein Expression and Purification—The Escherichia coli strain Rosetta2(DE3)pLysS cells transformed with the plasmids containing the DNA of Cyt c [13, 18] were inoculated in 5 ml of 2ϫTY medium and grown overnight. Two vinyl groups of heme c are linked via thioether bonds to two cysteines in Cyt c These linkages were fixed at crystallographic average bond distance values (see supplemental Table S1) to generate a reasonable complex structure for subsequent binding free energy calculations. ⌬Gpolar (⌬Gpolar, free energy for polar interactions) and ⌬Gnonpolar in the right-hand side of Equation 7 are the polar and nonpolar contributions to solvation, respectively The former term was calculated with the continuum solvation model based on the Poisson-Boltzmann equation [40], whereas the latter term was approximated by a simple linear relation for solvent-accessible surface area change (⌬ASA) [41]: ⌬Gnonpolar ϭ ␥ ⌬ASA ϩ , where ␥ ϭ 0.0072 kcalϪ1 molϪ1 A2 and  ϭ 0 [42]. Specific amino acid residues essential for the stabilization of the protein complex, in the binding interface of the Cyt c-CcO complex were identified using the free energy decomposition scheme with the MMPBSA.py script [43] implemented in AmberTools
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