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

The L-550 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 (SE) 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 L-550, a new SE signal is detected in the N-15 NMR spectrum which disappears upon thermal relaxation. This signal is in the range for a protonated SE, but downfield from the SE signals of bR(568) and N-520. Since steric interactions would have the opposite effect on shielding, the data argue against a 13,14-dicis chromophore in L-550. Comparison with the N-15 chemical shifts of halide salts of protonated Schiff bases (pSB's) of retinal suggests that the interaction of the SE with its counterion is significantly stronger in L-550 than in N-520 (which in rum is stronger than in bR(568)). This is consistent with models of the early photocycle in which the electrostatic interaction between the SE 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 lambda(max) = 491 nm of the chloride. This suggests some double bond strain in L-550, particularly about the C=N bond. This strain is apparently gone in the N intermediate, which has a normal relationship between the N-15 chemical shift and lambda(max). Such a relaxed chromophore is consistent with orientation of the SE proton toward the cytoplasmic surface in the N intermediate.