NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

Abstract

The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c ) to cytochrome c oxidase (C c O) is investigated by 1 H- 15 N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled C c O indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c , the specific line broadening of N-H signals disappeared and high field 1 H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with C c O are reduced by steric constraint in oxidized Cyt c , while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with C c O. These results suggest that the overall affinity of oxidized Cyt c for C c O is significantly, but not very much weaker than that of reduced Cyt c . Thus, electron transfer is gated by dissociation of oxidized Cyt c from C c O, the rate of which is controlled by the affinity of oxidized Cyt c to C c O for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon C c O binding to oxidized Cyt c , shown by 1 H- and 1 H, 15 N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.

Structural and Functional Analysis of Novel Human Cytochrome c Targets in Apoptosis

“…Interestingly, Cc uses the same surface surrounding the heme crevice (Fig. 4) to interact with all novel interaction partners as earlier studies have shown it to do with its physiological redox partners, namely, cytochrome c 1 (53), cytochrome-c peroxidase (54), and cytochrome-c oxidase (55) . Fig.…”

Respiratory complexes III and IV can each bind two molecules of cytochrome c at low ionic strength

“…These residues are surrounding the heme crevice and constitute the interaction surface (Fig. 2c), which is very well-conserved among complexes involving c-type cytochromes [14,29,[46][47][48][49][50][51][52][53][54][55]. In addition, significant CSP are detected (Dd avg -P 0.05 ppm) for Lys7, Lys13, Val20, Ser47, Lys73, Val83, Lys88 and Ala92.…”

“…Recently, the surface residues of hCc contacting bovine CcO have been mapped by NMR [52]. However, no evidences on the stoichiometry and binding affinity of the Cc-CcO complex were elucidated by NMR.…”

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

“…Recently, however, we successfully identified the interaction site for CcO on Cyt c using NMR spectroscopy (13). Our NMR analysis using chemical shift perturbations clearly indicated positively charged residues, including several Lys residues (Lys-5, Lys-7, Lys-8, Lys-13, Lys-79, Lys-86, Lys-87, and Lys-88), located in the interaction site for CcO on Cyt c, as we expected (10,14).…”

“…Our NMR analysis using chemical shift perturbations clearly indicated positively charged residues, including several Lys residues (Lys-5, Lys-7, Lys-8, Lys-13, Lys-79, Lys-86, Lys-87, and Lys-88), located in the interaction site for CcO on Cyt c, as we expected (10,14). In addition to the positively charged residues, several negatively charged residues such as Glu-4, Glu-89, Glu-90, and Asp-93 and some hydrophobic residues such as Ile-9, Ile-11, Met-12, and Ile-81 are also present in the interaction site for CcO, indicating that both electrostatic and hydrophobic interactions would contribute to ET complex formation between CcO and Cyt c, regulating the ET reaction from Cyt c to reduce molecular oxygen and promote proton pumping in CcO (13).…”