The absorption polarization of the first intermediate (K610) formed at room temperature in the proton-pumping photochemical cycle of bacteriorhodopsin (bR) shows a strong correlation with the polarization direction of the photolyzed parent molecule. The results suggest that, unlike other photosynthetic systems, excitation transfer does not take place prior to the primary photochemical change in bR. These observations together with the previously observed circular dichroism and the polarization temperature dependence are discussed in terms ofthe exciton structure and the nature of the absorption bandwidths (i.e., homogeneous vs. inhomogeneous) of the bR monomers within the trimer structure.Besides the chlorophyll-based systems, the only protein-pigment complex known to utilize light energy for biosynthetic purposes is bacteriorhodopsin (bR). The underlying molecular mechanism is intrinsically related to the cyclic photochemical behavior of bR, which is schematized as hp This cyclic photochemical process drives a proton pump providing a unidirectional proton translocation across the cell membrane (1-3). The vectorial proton movement results in the buildup of a proton gradient whose free energy is then directly available for ATP synthesis and other energy-requiring cellular processes. bR is composed of a relatively small, predominantly helical protein to which is attached either all-trans-retinal in the light-adapted (proton-pumping) state or a 1:1 mixture ofalltrans-and 13-cis-retinals when dark-adapted (4-6). In the native state, the chromoprotein molecules are highly immobilized in the bacterial membrane, forming a two-dimensional hexagonal lattice (7, 8) with the a-helical sequences of the protein oriented nearly perpendicularly to the membrane surface (9, 10). The pigment prosthetic group is probably covalently attached to the protein at a lysine residue near the inner membrane face (11)(12)(13) (21). Possible mechanisms involving both the ground and the excited-state properties are discussed, which might be responsible for the localization of the primary excitation on bR monomers and which are also consistent with the CD results (suggesting the existence of an exciton-type interaction). EXPERIMENTAL Purple membrane patches containing bR were extracted from a Halobacterium halobium strain R1 and purified by standard methods (25). The purified chromoprotein was suspended in doubly distilled water for measurements. The experimental arrangement is given in Fig. 1. Light-adapted samples with an optical density of 0.5 at 568 nm (e = 62.7 mM-1 cm-') were placed in a 1-cm rectangular fluorescence cuvette (Precision Cells, Hicksville, NY) with one of its faces perpendicular to the photolysis light beam. The sample was photolyzed by planepolarized 532-nm pulses with a 6-nsec half-width. The photo- The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abstract— The ultraviolet transient absorption assigned to the tyrosinate species in bacteriorhodopsin is followed in time and as a function of pH. Both its rise time and titration curve closely resemble those observed for the production of the M412 intermediate. These results may support a recently proposed mechanism that couples tyrosinate production to the Schiff base deprotonation in the proton pump of bacteriorhodopsin.
The photolysis-induced changes in the protein fluorescence intensity (at 320 nm) during the proton-pumping cycle of bacteriorhodopsin were examined by a delayed two-pulse technique in the time range 1 psec-20 msec at room temperature. No detectable change in the protein fluorescence intensity was observed on the earliest time scale within the lifetime of the intermediate K when retinal apparently undergoes the largest structural changes. The time dependence of the relative changes in fluorescence intensity did, however, display a close correlation with the population of the LM and M412 intermediates. From a computer numerical fit of the data, with available published kinetic parameters, the protein fluorescence quantum yields of the K590, L I and M412 intermediates are found to be 1.0, 0.92, and 0.80 of that for native bR570, respectively. The probable mechanisms of the observed fluorescence quenching during the photochemical cycle are qualitatively discussed.Most work done on bacteriorhodopsin (bR) has focused attention mainly on the role of the Schiff-base-linked retinal (1, 2). Less is known about the involvement of the protein moiety in the photochemical cycle and related processes, although the structural-functional unit of retinal and bacterio-opsin is evident. Ultrafast spectroscopic results (3, 4) suggest that the protein may respond almost instantaneously with a rapid protontransfer reaction to photon absorption by the chromophore; numerous studies (5-19) additionally indicate that the protein undergoes a series ofconformational changes as the photochemical process proceeds. However, only moderate success has been achieved in identifying molecular motions within the apoprotein and in correlating them with the well-characterized photochemical intermediates. The results of the research done on this problem (10-13, 17-19) disagree as to the origins and the extent ofthe aromatic amino acid perturbations. Changes in the intrinsic protein fluorescence ofbR, although small, seem to offer promising opportunities to monitor the time-dependent interactions (involving tyrosine and tryptophan residues) within the chromoprotein during the photochemical cycle. This communication reports on some correlations between fluorescence intensity changes and the populations of the photochemical intermediates in the microsecond and millisecond time domains.MATERIALS AND METHODS Carotenoid-free bR was isolated and purified by the method of Becher and Cassim (20) and used without sonication. Suspensions were prepared in doubly distilled water at a concentration of 22 AM + 10% (e = 63,000 liters/mol cm) in all experiments.Samples placed in a semimicro quartz absorption cuvette with a 1.0-cm optical pathlength (Markson, type E-18, Del Mar, CA) were light adapted prior to fluorescence measurements by exposure to standard room light for at least 2 hr. To minimize the influence of polarization effects (the possible excitation of populations with different polarization properties) we illuminated the sample through one of the ...
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