Amino Acid Substitutions in the Non-Ordered Ω-Loop 70–85 Affect Electron Transfer Function and Secondary Structure of Mitochondrial Cytochrome c

Rita V Chertkova,Alexey R Brazhe,Victor V Revin,Nadezhda A Brazhe,Tatyana V Bryantseva,Alexei N Nekrasov,Kseniya S Kudryashova,Andrew B Rubin,Alexander I Yusipovich,Dmitry A Dolgikh,Mikhail P Kirpichnikov,Georgy V Maksimov

doi:10.3390/cryst11080973

Rita V Chertkova, Alexey R Brazhe + Show 10 more

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https://doi.org/10.3390/cryst11080973

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Abstract

The secondary structure of horse cytochrome c with mutations in the P76GTKMIFA83 site of the Ω-loop, exhibiting reduced efficiency of electron transfer, were studied. CD spectroscopy studies showed that the ordering of mutant structure increases by 3–6% compared to that of the WT molecules due to the higher content of β-structural elements. The IR spectroscopy data are consistent with the CD results and demonstrate that some α-helical elements change into β-structures, and the amount of the non-structured elements is decreased. The analysis of the 1H-NMR spectra demonstrated that cytochrome c mutants have a well-determined secondary structure with some specific features related to changes in the heme microenvironment. The observed changes in the structure of cytochrome c mutants are likely to be responsible for the decrease in the conformational mobility of the P76GTKMIFA83 sequence carrying mutations and for the decline in succinate:cytochrome c-reductase and cytochrome c-oxidase activities in the mitoplast system in the presence of these cytochromes c. We suggest that the decreased efficiency of the electron transfer of the studied cytochromes c may arise due to: (1) the change in the protein conformation in sites responsible for the interaction of cytochrome c with complexes III and IV and (2) the change in the heme conformation that deteriorates its optimal orientation towards donor and acceptor in complexes III and IV therefore slows down electron transfer. The results obtained are consistent with the previously proposed model of mitochondrial cytochrome c functioning associated with the deterministic mobility of protein globule parts.

Highlights

This article is an open access articleCytochrome c is known to be involved in electron transfer from ubiquinol–cytochrome c-oxidoreductase to cytochrome c-oxidase in the mitochondrial respiratory chain
We obtained a series of additional mutant variants, with substitutions aimed at a greater decrease in the electron transport activity, in particular, cytochrome c-oxidase activity, as the final stage of electron transfer in the electron transport chain (ETC)
The structure of cytochrome c is a dynamic system consisting mainly of α-helices. Parts of this dynamic system are rearranged during the functioning of the protein, and conformation changes occur in the loop regions

Summary

Introduction

This article is an open access articleCytochrome c is known to be involved in electron transfer from ubiquinol–cytochrome c-oxidoreductase (complex III) to cytochrome c-oxidase (complex IV) in the mitochondrial respiratory chain. Type c heme (hematoporphyrin) is the prosthetic group in cytochrome c that ensures its electron transfer function [1]. IR spectroscopy studies proved that this transition is accompanied by rearrangements of some hydrogen bonds within the heme and diffusion of intramolecular water, as well as by changes in the ratio between the fraction of β-structural elements and α-helices [4,5,6]. Changes in the spatial structure of the protein are realized mainly in weakly structured and looped areas of the globule. The Ω-loops play a crucial role in structural rearrangements that precede electron transfer in the cytochrome c heme and other electron transport proteins or oxygen binding in hemoglobin and myoglobin [7,8,9,10]. Cytochrome c was found to contain three Ω-loop regions corresponding to residues 20–35, 36–61, and

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