Interaction of cytochrome c with NO studied by time-resolved raman and absorption spectroscopy

Abstract : Recently, it has been shown that cytochrome c (cyt c) released during apoptosis is nitrosylated in mitochondria, the first case of its nitrosylation in vivo (Schonhoff, C. M., Gaston, B., and Mannick, J. B. (2003) J. Biol. Chem., 278, 18265). In native cyt c, the heme-iron is ligated to the protein through two axial bonds, involving histidine and methionine. The nitrosylation of cyt c during apoptosis led to the hypothesis of its activation due to nitric oxide (NO) binding which would induce a structural change of cyt c. Similarly to guanylate cyclase this conformational change may be triggered by the breaking of Fe2 +-methionine bond. For NO binding to occur, the cleavage of the Fe-methionine bond must transiently occur. Consequently, an equilibrium between the following forms should exist with a low dissociation energy of this bond: [Met - Fe - His] <- ->[Met Fe- His] - NO -> [Met NO - Fe - His] This hypothesis is strengthened by the observation that the Fe -Met bond is cleaved already upon a small temperature increase. To investigate the properties of the transient species involved in this scheme, we used flash photolysis and ultrafast detection techniques to generate and characterize the relevant 5-coordinate species. We characterized the ferric and ferrous cyt c NO adducts by steady state Raman spectroscopy and we used ultrafast time-resolved absorption and Raman spectroscopy. TRRR allows to measure the evolution of vibrational frequencies related to structural changes with a time resolution of 0.6 10 12 s. After photoexcitation of nitrosylated ferrous cyt c we observed two relaxation times (t 1 = 2 ps and t 2 = 8 ps) attributed respectively to the vibrational relaxation of the five-coordinate hot species and to the recombination of the NO ligand. These times are of the same order of magnitude as we observe for recombination of methionine ligand in native ferrous cyt c with different relaxation times (t1 = 1.8 ps and t2 = 5.5 ps). The time dependence of Raman spectra of ferrous cyt c and ferrous cyt c NO adducts compared with the stationary spectra show clearly that a conformational change is induced during the ligand release in the case of nitrosylated ferrous cyt c with respect to what observed for native ferrous cyt c. Correlation between this conformational change and the biological activity of the protein will be discussed.
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S. Cianetti, Sergei Kruglik, Marten Vos, P. Y. Turpin, Jean-Louis Martin, et al.. Interaction of cytochrome c with NO studied by time-resolved raman and absorption spectroscopy. 13th EUROPEAN BIOENERGETICS CONFERENCE, Aug 2004, Pisa, Italy. pp.218. ⟨hal-00845030⟩

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