Bacteriorhodopsin Intermediate Spectra Determined over a Wide pH Range Csilla Gergely,* La ´ szlo ´ Zima ´ nyi, and Gyo 1 rgy Va ´ ro ´ Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary ReceiVed: April 24, 1997 X The pH dependence of the absorption spectra of bacteriorhodopsin and its photocycle intermediates was studied in the pH range 4.5-9. The spectra of the intermediates were determined from difference spectra taken during the photocycle with an optical multichannel analyzer. The data analysis was based on various criteria concerning the shape of the spectra, but no assumption was made about the kinetic model that describes the photocycle. The strategy for calculation of the spectra was one described earlier, as well as a newly introduced algorithm based on the Monte Carlo method. The search methods used gave very similar results. Like the absorption spectrum of bacteriorhodopsin in the above-mentioned pH range, the spectra of all the intermediates were found to be almost unchanged. The spectrum of intermediate M displayed a 2 nm, and that of intermediate L a 1 nm, red shift with rising pH, but this latter shift was within the overall error of the measurements. The similarity of all the intermediate spectra calculated with different procedures at all pH values points to the reliability of the method and the validity of the spectra. Averaging of the calculated spectra over the whole pH range furnished a well-determined set of intermediate spectra, suitable for further kinetic and spectroscopic studies of the photocycle. Introduction Bacteriorhodopsin (BR), the only protein in the purple membrane of Halobacterium salinarum, functions as a light- driven proton pump. 1-3 Its structure is known with 0.35 nm resolution. 4,5 Upon absorbing light the retinal chromophore of BR passes through an excited state to a higher-energy state, and a series of thermal reactions drive the protein through several spectrally distinct intermediates (denoted K, L, M, N, and O) back to the initial form, BR. Most of the intermediates have been decomposed kinetically to several spectroscopically indistinguishable substates. Recent reviews describe the details of the photocycle and the related proton pumping mechanism. 3,6,7 The pH dependence of the BR spectrum has been investigated intensively. When the pH of the purple membrane suspension is mentioned, it must be borne in mind that it is usually measured in the bulk, but, depending on the ionic strength of the solution, the pH at the membrane surface will be lower due to the negative surface charge. When the ion concentration of the suspension is lowered, this difference increases and can reach several pH units. 8 In the pH interval 4-9 at moderate ionic strength (several hundred mM), where the surface pH is presumed to be approximately equal to that measured in the bulk, the spectra of the ground-state BR are considered practically unchanged. 9,10 At pH below 4, BR passes through a series of transformations, depending on the nature and concentration of the ions in the suspension. 11-13 One very pronounced change is the transition from purple to blue membrane with a pK a 2.5 when no chloride is present. 13 At pH above 9, the deprotonation of some amino acids produces a smaller spectral change, 12,14,15 and at even higher pH the Schiff base can deprotonate without illumination. Recent findings show that there is a spectral change with pK a around 9.7, a secondary transition of the blue to purple membrane, which can be observed only at long wavelengths, around 700 nm. The explanation of the two pK a ’s is based on the complex titration of residue D85 together with another proton acceptor group in the extracellular half-channel of BR. 16 The first attempts to determine the spectra of the photocycle intermediates were performed by trapping the BR sample in different intermediate states at low temperature. 17,18 With the introduction of the optical multichannel analyzer, the possibility arose for the detection of the whole spectral change at a given time after excitation and for calculation of the spectra of the intermediates. 19-21 Investigations of the effect of pH upon the BR photocycle (for a review, see ref 7) assume that the spectra of the intermediates are pH-independent, 21,22 but the validity of this hypothesis has never been proven. In the present work, this question was investigated in the pH range from 4.5 to 9, where the spectrum of BR can be considered to be unchanged. The spectra of the intermediates were calculated without any assumption concerning the kinetic model of the photocycle, but several hypotheses regarding the shape of the spectra were used: non-negative absorption, smooth spectra, only one main peak, and shapes resembling the skewed spectra of the rhodo- psin-type pigments. 23-25 The spectra were unchanged or only slightly affected by the pH change. The spectrum of intermedi- ate M shifted toward the red by about 2 nm and that of L intermediate by about 1 nm in the same direction as the pH was raised, but the latter shift was within the overall error of the analysis. Materials and Methods Purple membranes were isolated from H. salinarium strain Sg by the standard procedure described previously. 26 The isolated purple membranes were embedded in polyacrylamide gel, by means of a method reported elsewhere, 11 having a BR concentration of about 15 μM. The gel was soaked overnight in 100 mM NaCl, 25 mM Bis-Tris propane, and 25 mM phosphate buffers at the desired pH. It was light-adapted prior to the measurement and thermostated at 20 °C during data collection. Absorption spectra of the light-adapted BR contain- * Corresponding author. E-mail: Gergelycs@everx.szbk.u-szeged.hu. Fax: 36-62-433133. X Abstract published in AdVance ACS Abstracts, October 15, 1997. 9390 J. Phys. Chem. B 1997, 101, 9390-9395 S1089-5647(97)01381-3 CCC: $14.00 © 1997 American Chemical Society Downloaded by UNIV OF GUELPH LIBRARY on October 22, 2009 | http://pubs.acs.org Publication Date (Web): November 6, 1997 | doi: 10.1021/jp971381e