Electrogenic Proton-Pumping Capabilities of the M-Fast and M-Slow Photocycles of Bacteriorhodopsin

Abstract

The parallel model for the bacteriorhodopsin (BR) photocycle at neutral pH and a temperature near 20 °C contains an M-fast cycle with steps BR → K → L → M f → N → O → BR and an M-slow cycle which contains steps BR → K → L → M s → BR. With increasing actinic laser strength, the M-fast cycle at first rises faster than the M-slow cycle, but reaches saturation sooner and at a lower level than the M-slow cycle. The O-intermediate shows the same saturation behavior as M f . In this paper, we show that the peak current of proton flux and the apparent voltages developed by this flux show the same saturation behavior as M s , which is very different from that of both M f and O. It is further shown that most of the proton-charge displacement is connected with the step M s → BR. The optical and electrical data in these studies were collected simultaneously by a newly designed and built spectrometer which is described separately.Kinetic analyses of bacteriorhodopsin (BR 1 ) photocycle data have led to two distinctly different kinds of models (1). In one there is a single photocycle with reversible steps emanating from a homogeneous ground state, while the other has two or more independent photocycles originating from different forms of BR. While the single photocycle model is much more widely accepted, our laboratory has long been a subscriber to a model involving parallel photocycles. In a recent publication (2), the evidence for both kinds of models was presented in some detail, as well as the reasons for our favoring the parallel photocycle model.The parallel photocycle for BR at pH near 7 and temperature near 20 °C consists of two separate cycles, one containing M f and the other M s . † This research was supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute. ‡ Certain commercial equipment, instruments, or materials are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institutes of Health or the National Institute of Standards and Technology, or that the materials and equipment are necessarily the best available for the purpose. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 May 11. Published in final edited form as:Biochemistry. where K, L, M, N, and O are the recognized intermediates of the BR photocycle and the subscripts "f" and "s" indicate the faster and slower time constants for intermediates displaying the same characteristic spectra. The maximum characteristic absorbance wavelengths for these individual spectra are listed in Using the procedures of Kesthelyi and Dér and their collaborators (4,5), one can measure the proton current which flows during a single turnover of BR. This is accomplished by orienting the purple membrane (PM) in an electric field in an optical cuvette and holding this orientation by forming an acyrlamide gel of the oriented population. The current is measured using two platinized electrodes and a suitable amplifi...