Time-resolved absorption and photothermal measurements with sensory rhodopsin I from Halobacterium salinarum

An expansion accompanying the formation of the first intermediate in the ph otocycle of transducer-free sensory rhodopsin I (SRI) was determined by mea ns of time-resolved laser-induced optoacoustic spectroscopy. For the native protein (SRI-WT), the absolute value of the expansion is similar to 5.5 mL and for the mutant SRI-D76N, similar to 1.5 mL per mot of phototransformed species (in 0.5 M NaCl), calculated by using the formation quantum yield f or the first intermediate (S-610) of Phi(610) = 0.4 +/- 0.05 for SRI-WT and 0.5 +/- 0.05 for SRI-D76N, measured by laser-induced optoacoustic spectros copy and by laser flash photolysis. The similarity in Phi(610) and in the d etermined value of the energy level of S-610, E-610 = (142 +/- 12) kJ/mol f or SRI-WT and SRI-D76N indicates that Asp(76) is not directly involved in t he first step of the phototransformation, The increase with pH of the magni tude of the structural volume change for the formation of S-610 in SRI-WT a nd in SRI-D76N upon excitation with 580 nm indicates also that amino acids other than Asp(76), and other than those related to the Schiff base, are in volved in the process. The difference in structural volume changes as well as differences in the activation parameters for the S-610 decay should be a ttributed to differences in the rigidity of the cavity surrounding the chro mophore, Except for the decay of the first intermediate, which is faster th an in the SRI-transducer complex, the rate constants of the photocycle for transducer-free SRI in detergent suspension are strongly retarded with resp ect to wild-type membranes (this comparison should be done with great care because the preparation of both samples is very different).