Optoacoustic Studies of Electron Transfer Processes in Modified Proteins

PEDRO M. DAVID GARA , GABRIEL M. BILMES , LIONEL CHERUZEL AND SILVIA E. BRASLAVSKY

Congreso; 16th International Congress on Photobiology.; 2014

The long-range (10 to 25 Å) charge transfer process in protein complexes is a fundamental process in respiratory and photosynthetic machinery. There are several open questions about the contributions of the environment in these long-range interactions as well as about the influence of the separation between the donor (D) and acceptor (A) partners. In this work the structural movements in proteins due to charge transfer are studied by laser-induced photoacoustic measurements. The systems studied are 2 types of proteins: one is the CuA centres of cytochrome C oxidase subunit II [1]. The other consist in two mutants of cytochrome P450 (K97C and Q397C) with a Ru(II)(bpy)2(IA-phen) complex acting as electron donor upon excitation, attached at different positions to the protein [2,3]. Deconvolution methods for signal analysis, in combination with an appropriate model were used for the determination of structural volume changes. Three well-separated processes were identified: a fast one (prompt) with a decay time 1 ≤ 30 ns; a slower process with a lifetime 2 ca. 500-600 ns, and a longest-lived component with a lifetime longer than 1 s. The pre-exponential factors φi of the three components at several temperatures were plotted as E φi vs (cp/β). In all cases, good linear correlations were obtained. The slopes of the lines represent the structural volume change associated with each of the processes, in each case multiplied by the respective quantum yield of the process. The scheme shows the possible processes generated after laser excitation for cytochrome P450 BM3 K97C. The intermediate I1 can be assigned to excited Ru in the protein (*RuII K97C-FeIII P45), I2 is assumed to be the metal to ligand charge transfer state of the Ru(bpy)2(IA-phen), and I3 can be attributed to electron transfer between the two metal centres (RuIII K97C-FeIII P450). Scheme 1: possible processes generated after laser excitation of P450 BM3 K97C Acknowledgments authors acknowledge Dr. A. Vila and Dr. L. Abriata from Instituto de Biologia Molecular y Celular de Rosario, for the Citocrome oxidase samples. [1] L. A. Abriata, G. N. Ledesma, R. Pierattelli, and A. J. Vila. J. Am. Chem. Soc. 2009, 131, 1939. [2] M. E. Ener, Y.-T. Lee, J. R. Winkler, H. B. Gray, and L. Cheruzel. PNAS, 2010, 107, 18783. [3] N.-H. Tran, N. Huynh, T. Bui, Y. Nguyen, P. Huynh, M. E. Cooper and L. E. Cheruzel. Chem. Commun., 2011, 47, 11936.