Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

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.

Keywords: bioenergetics; cytochrome c; cytochrome c oxidase (complex IV); electron transfer complex; molecular docking.

FIGURE 1.

Molecular representation of human Cyt c and bovine CcO. a, structure of human Cyt c (red schematic) with the residues (spheres) targeted by in vitro mutagenesis. In detail, the mutated residues in groups A–C, reflecting the degree of mutational effects (severe, moderate, and unaffected, respectively), are represented by magenta, cyan, and green spheres, respectively. b, structure of bovine CcO with the three important catalytic subunits I–III represented by green, cyan, and magenta, respectively. c, blue region of CcO used for docking includes the part (partial residues in subunits I–IV and VII–XIII) occupying the intermembrane space and the adjacent membrane-embedded region. In CcO, heme groups and copper ions are represented in spheres.

FIGURE 2.

Absorbance change at 550 nm after the addition of CcO (a) and the dependence of the initial rate constants on the concentrations of ferrous Cyt c (b). a, small amount of potassium ferricyanide(III) was added to the reaction mixture (downward arrow) to determine the end point of the ET reaction. The measurements were carried out in the presence of 1 nm CcO dissolved in 50 mm sodium phosphate buffer at pH 6.8 containing 0.1% n-decyl β-d-maltoside. b, solid curve is the best fit to the Michaelis-Menten equation using the least-square analysis.

FIGURE 3.

Predicted binding interfaces of the Cyt c-CcO complexes of poses 1 and 13. a, focused view of the heme c (sphere) in Cyt c and Trp-104 (stick) in CcO, the probable electron entry site obtained from pose 1. b, interactions of the mutated residues in Cyt c with contact residues in CcO from pose 1 are highlighted. The amino acid residues in subunits I, II, and III are represented by green, cyan, and magenta (sticks), respectively. Dotted lines indicate hydrogen bonding between Cyt c and CcO residues. c and d, same representation as in a and b, respectively, but the corresponding CcO is from pose 13.

FIGURE 4.

Plots of ΔG_obs with ΔG_calc. The calculated values are obtained from the MM-PBSA calculation on the wild-type and mutant complexes constructed from pose 1 (a) and pose 13 (b).The correlation equation (ΔG_calc = 0.0548 (ΔE_coul + ΔE_vdW + ΔG_polar + ΔG_nonpolar)− 0.899) was used for the calculation.

FIGURE 5.

a, free energy decomposition of ΔG_tot on a per residue basis into the contributions from ΔE_vdW, ΔG_electro, and ΔG_nonpolar (|ΔG_tot| ≥ 3.0 kcal mol⁻¹). Three-dimensional views of the residues at the regions of whole interface (b), subunit I (c), subunit II (d), and subunits III–XIII (e) other mainly contributing to the binding of Cyt c to CcO.