Amino Acid Substitutions in the Non-Ordered Ω-Loop 70–85 Affect Electron Transfer Function and Secondary Structure of Mitochondrial Cytochrome <i>c</i>
Rita V. Chertkova,
Tatyana V. Bryantseva,
Nadezhda A. Brazhe,
Kseniya S. Kudryashova,
Victor V. Revin,
Alexei N. Nekrasov,
Alexander I. Yusipovich,
Alexey R. Brazhe,
Andrew B. Rubin,
Dmitry A. Dolgikh,
Mikhail P. Kirpichnikov,
Georgy V. Maksimov
Affiliations
Rita V. Chertkova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Tatyana V. Bryantseva
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Nadezhda A. Brazhe
Biophysics Department, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory, 1/12, 119234 Moscow, Russia
Kseniya S. Kudryashova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Victor V. Revin
Department of Biotechnology, Bioengineering and Biochemistry, National Research Ogarev Mordovia State University, 430005 Saransk, Russia
Alexei N. Nekrasov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Alexander I. Yusipovich
Biophysics Department, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory, 1/12, 119234 Moscow, Russia
Alexey R. Brazhe
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Andrew B. Rubin
Biophysics Department, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory, 1/12, 119234 Moscow, Russia
Dmitry A. Dolgikh
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Mikhail P. Kirpichnikov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, 117997 Moscow, Russia
Georgy V. Maksimov
Biophysics Department, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory, 1/12, 119234 Moscow, Russia
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.
