Characterization of the branched-photocycle intermediates P and Q of bacteriorhodopsin

The bacteriorhodopsin branched-photocycle intermediates P and Q are studied with respect to chromophore isomeric content, photochemical origin, kinetic heterogeneity, and photoreversibility. These blue-shifted species are compared to products with similar spectroscopic properties generated via thermal denaturation. We observe that the thermal and photochemical species differ in both isomeric content and binding site environment. Sequential two-photon activation of glycerol suspensions of bacteriorhodopsin containing low concentrations of water (<15% v/v water/glycerol) form high yields of P state but almost no Q state. This observation is attributed to the role that water plays in the hydrolysis reaction that converts the P state to Q. Relatively large photostationary state populations of both intermediates can be generated in both 85% v/v glycerol suspensions and polyacrylamide gels. At ambient temperature, both intermediates can be fully converted back to bR with blue light. Chromophore extraction and HPLC analysis reveal that P, Q, and the spectrally similar thermal products have a 9-cis retinal chromophore. The thermal products are also found to contain small amounts of the 7-cis isomer. Time-resolved spectroscopy reveals that the P state is actually comprised of two components with maximum absorptivities at 445 and 525 nm. The molar absorptivities of the chromophore band maxima of the P and Q states in an 85% v/v glycerol/water suspension at pH 7 are epsilon(P-445)=47000, epsilon(P-525)=39000, and epsilon(Q)=33000 M-1 cm(-1). Kinetic analyses support previous studies indicating that the P state is formed predominantly from the 0 state, which is consistent with the branched-photocycle model. However, other paths to the P state are possible, including direct excitation of the small population of blue membrane present in solution. We examine the hypothesis that the branched-photocycle evolved as a photochromic sunscreen for UVA and UVB protection.