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 dr awn much attention with respect to the mechanism of light-driven proto n transport because it selectively releases the Schiff base (SE) proto n 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 relax ation. 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 wo uld have the opposite effect on shielding, the data argue against a 13 ,14-dicis chromophore in L-550. Comparison with the N-15 chemical shif ts of halide salts of protonated Schiff bases (pSB's) of retinal sugge sts that the interaction of the SE with its counterion is significantl y 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 whic h the electrostatic interaction between the SE and its counterion cons titutes an important constraint. Although the L counterion interaction is comparable to that of a 6-s-trans,13-cis chloride salt, the visibl e spectrum is strongly red-shifted from the lambda(max) = 491 nm of th e chloride. This suggests some double bond strain in L-550, particular ly about the C=N bond. This strain is apparently gone in the N interme diate, which has a normal relationship between the N-15 chemical shift and lambda(max). Such a relaxed chromophore is consistent with orient ation of the SE proton toward the cytoplasmic surface in the N interme diate.