Primary step in the bacteriorhodopsin photocycle: photochemistry or excitation transfer?

Abstract

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.

Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin

“…In over a decade and a half an impressive body of experimental and theoretical evidence has been accumulated consistent with an exciton model of interactions among the retinylidene rrr* induced transition dipole moments of the bR's in the PM (Becher and Cassim, 1975a;Heyn et al, 1975Heyn et al, , 1977aBauer et al, 1976;Becher and Ebrey, 1976;Kriebel and Albrecht, 1976;Cherry et al, 1977Cherry et al, , 1978Ebrey et al, 1977;Dencher and Heyn, 1978;Muccio and Cassim, 1979a, b;Casadio and Stoeckemius, 1980;Papadopoulos and Cassim, 1981a;Klausner et al, 1982;Draheim and Cassim, 1985a,b;Draheim et al, 1988a). Recently, El-Sayed and co-workers have discussed a number of experimental observations which they claim could cast serious doubts on the viability of this exciton model of the PM (El-Sayed et al, 1981;El-Sayed et al, 1989;Jang et al, 1990). These observations are (a) fluorescence and absorption of the K,,0 photointermediate of the bR are highly polarized with respect to the initial polarized excitation, (b) absence of a biphasic magnetic circular dichroism (MCD) spectrum for bR5., (c) KY6,and L550 photointermediates, in which the trimeric structure is destroyed by the isomerization of one of the three retinals, exhibit CD spectra similar to that of bRS68 in 58-65% glycerol solutions based on a study of Zimainyi et al (1987), and (d) the mutagenic substitution of Tyn (185) by Phe is found to greatly change the CD in Y185F as compared with eBR.…”

CD spectrum of bacteriorhodopsin

“…These observations are summarized as following: (a) If exciton coupling is present, one would expect rapid energy transfer of the excitation between the different retinals in bR which would lead to depolarized retinal absorption for the first daughter formed during the photocycle of polarized excitation. However, the absorption of the K6,0 (25,26) are found to be highly polarized with respect to the initial polarized excitation. (b) The absence of biphasic MCD spectrum for bR (11) indicates the absence of the doubly degenerate state predicted by the exciton model (17).…”

“…(3) This time is much shorter than the formation time of the first photochemical intermediate in bR photocycle, which is found to be 400 fs (30,31). It is thus difficult to explain the observed high anisotropy of the linear dichroism of the retinal absorption in the K intermediate (25,26) FIGURE 6 Plotting of CD spectrum (O/v) versus the frequency v for completely reduced bR sample. The spectrum is used to calculate the value of the rotational strength (see Table 1).…”

Comparison of the Dynamics of the Primary Events of Bacteriorhodopsin in Its Trimeric and Monomeric States

“…As a light-driven proton pump, bR is the other type of photosynthetic center besides chlorophyll. Since its discovery, bR has served as the simplest model for studying bioenergetics for more than three decades (El-Sayed et al, 1981;Birge, 1981;Mathies et al, 1991;Ebrey, 1993;Lanyi, 1997).…”