Steric barrier to bathorhodopsin decay in 5-demethyl and mesityl analoguesof rhodopsin

Absorbance difference spectra were recorded from 20 ns to 1 mus after 20 T photoexcitation of artificial visual pigments derived either from 5-demethy lretinal or from a mesityl analogue of retinal. Both pigments produced an e arly photointermediate similar to bovine bathorhodopsin (Batho). In both ca ses the Batho analogue decayed to a lumirhodopsin (Lumi) analogue via a blu e-shifted intermediate, BSI, which formed an equilibrium with the Batho ana logue. The stability of 5-demethyl Batho, even though the C8-hydrogen of th e polyene chain cannot interact with a ring C5-methyl group to provide a ba rrier to Batho decay, raises the possibility that the 5-demethylretinal rin g binds oppositely from normal to form a pigment with a 6-s-trans ring-chai n conformation. If 6-s-trans binding occurred, the ring Cl-methyls could re place the C5-methyl in its interaction with the chain C8-hydrogen to preser ve the steric barrier to Batho decay, consistent with the kinetic results. The possibility of 6-s-trans binding for 5-demethylretinal also could accou nt for the unexpected blue shift of 5-demethyl visual pigments and could ex plain why 5-demethyl artificial pigments regenerate so slowly. Although the mesityl analogue BSI's absorption spectrum was blue-shifted relative to it s pigment spectrum, the blue shift was much smaller than for rhodopsin's or 5-demethylisorhodopsin's BSI. This suggests that increased C6-C7 torsion m ay be responsible for some of BSI's blue shift, which is not the case for m esityl analogue BSI either because of reduced spectral sensitivity to C6-C7 torsion or because the symmetry of the mesityl retinal analogue precludes having 6-s-cis and 6-s-trans conformers. The similarity of the mesityl anal ogue BSI and native BSI pi (max) values supports the idea that BSI has a 6- s angle near 90 degrees, a condition which could disconnect the chain (and BSI's spectrum) from the double bond specifics of the ring.