The predischarge chromophore in bacteriorhodopsin: a 15N solid-state NMR study of the L photointermediate

The L550 intermediate in the bacteriorhodopsin (bR) photocycle has drawn much attention with respect to the mechanism of light-driven proton transport because it selectively releases the Schiff base (SB) proton to the extracellular proton channel in the L-->M transition. Here we extend our solid-state NMR studies of bR photocycle intermediates to the L state. Under conditions that stabilize L550, a new SB signal is detected in the 15N NMR spectrum which disappears upon thermal relaxation. This signal is in the range for a protonated SB, but downfield from the SB signals of bR568 and N520. Since steric interactions would have the opposite effect on shielding, the data argue against a 13,14-dicis chromophore in L550. Comparison with the 15N chemical shifts of halide salts of protonated Schiff bases (pSB's) of retinal suggests that the interaction of the SB with its counterion is significantly stronger in L550 than in N520 (which in turn is stronger than in bR568). This is consistent with models of the early photocycle in which the electrostatic interaction between the SB and its counterion constitutes an important constraint. Although the L counterion interaction is comparable to that of a 6-s-trans,13-cis chloride salt, the visible spectrum is strongly red-shifted from the lambdamax = 491 nm of the chloride. This suggests some double bond strain in L550, particularly about the C=N bond. This strain is apparently gone in the N intermediate, which has a normal relationship between the 15N chemical shift and lambdamax. Such a relaxed chromophore is consistent with orientation of the SB proton toward the cytoplasmic surface in the N intermediate.