Time-Resolved Spectroscopy of the Early Photolysis Intermediates of Rhodopsin Schiff Base Counterion Mutants

Abstract

Time-resolved absorption difference spectra of COS-cell expressed rhodopsin and rhodopsin mutants (E113D, E113A/A117E, and G90D), solubilized in detergent, were collected from 20 ns to 510 ms after laser photolysis with 7 ns pulses (λ_max = 477 nm). The data were analyzed using a global exponential fitting procedure following singular value decomposition (SVD). Over the entire time range excellent agreement was achieved between results for COS-cell and rod outer segment rhodopsin both in kinetics and in the λ_max values of the intermediates. The Schiff base counterion mutant E113D showed strong similarities to rhodopsin up to lumi, following the established scheme: batho ⇌ bsi → lumi. Including late delay times (past 1 μs), the mutant E113D lumi decayed to metarhodopsin II (MII), showing that the detergent strongly favors MII over metarhodopsin I (MI). However, a back-reaction from MII to lumi was observed that was not seen for rhodopsin. The kinetic schemes for the mutants E113A/A117E and G90D were significantly different from that of rhodopsin. In both mutants batho decay into an equilibrium with bsi was too fast to resolve (12−C₁₃ of the retinal chromophore. The λ_max values of the bsi and lumi intermediates in the mutant pigments are interpreted in terms of movement of the Schiff base relative to its counterion.