Mechanism of Activation of Cytochrome c Peroxidase Activity by Cardiolipin

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Yu. A. Vladimirov^1*, E. V. Proskurnina¹, D. Yu. Izmailov¹, A. A. Novikov², A. V. Brusnichkin³, A. N. Osipov², and V. E. Kagan⁴

¹Faculty of Fundamental Medicine, Lomonosov Moscow State University, Lomonosovskii pr. 31/5, 117192 Moscow, Russia; fax: (495) 932-8814; E-mail: yuvlad@mail.ru

²Russian State Medical University, ul. Ostrovityanova 1, 117997 Moscow, Russia; E-mail: anosipov@yahoo.com

³Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; E-mail: x6f.rebel@rambler.ru

⁴University of Pittsburgh, 100 Technology Dr., Suite 333, 15260 Pittsburgh, PA, USA; E-mail: vkagan@ceoh.pitt.edu

^* To whom correspondence should be addressed.

Received January 10, 2006; Revision received March 29, 2006

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In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe···S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695-700 nm (A₆₉₅) were compared. A strict concurrency between Fe···S(Met80) breaking (A₆₉₅) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0 : 1 to 50 : 1 (by chemiluminescence). Nevertheless, when A₆₉₅ completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (=4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16-20-fold. A₆₉₅ did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1 : 10 : 10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe···S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe···S(Met80) breaking as the result of conformational changes in the protein globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H₂O₂ as a result of the hydrophobic interaction between cardiolipin and cytochrome.

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KEY WORDS: cytochrome c, cardiolipin, peroxidase activity

DOI: 10.1134/S0006297906090070

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