Photoexcitation of the O intermediate of bacteriorhodopsin and its mutant E204Q

The light-driven proton pump, bacteriorhodopsin (bR), in Halobacterium salinarum carries out its function proceeding through intermediates named J, K, L, M, N, and O (for recent reviews see [1, 2]). To understand the molecular mechanism of light-energy transduction many methods are used [3]. One of them, photoexcitation of the intermediates, provides valuable information on their particular rôle in the photocycle [4]. Electric responses of the photoreactions of the intermediates turned out to be indispensable in understanding the mechanism of proton translocation. It became clear that illumination of the M intermediate with blue light terminated proton transfer [5]. In time-resolved studies negative currents were registered [6, 7] (negative current is defined relative to the positive direction of undisturbed proton transport). This negative photoelectric response helped to verify the existence of a very long living M intermediate in the photocycle of the bR mutant D96N [8]. In a recent paper we published data on the photoelectric responses of the N intermediate [9]. The O intermediate has been rather elusive in the case of wild-type bR (WTbR). Later, it became clear that it accumulates at low pH and high temperature [10, 11]. The mutant E204Q has a long living O even at normal pH and room temperature [12, 13]. The study of the photoelectric responses of O, accumulating during the photocycle of WTbR and E204Q, is of special interest not only because electric responses from O excitation are less known but also because the late intermediates are still in the focus of research [11]. Performing double flash excitation we found quite different photoreactions for O of WTbR and E204Q: a pumping photocycle occurs for WTbR and a small disturbance for the mutant E204Q.