Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy

Time-resolved resonance Raman microchip flow experiments have been performe d on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediat es of rhodopsin at room temperature to elucidate the structure of the chrom ophore in each species as well as changes in protein-chromophore interactio ns. Transient Raman spectra of Lumi and Meta I with delay times of 16 mus a nd 1 ms, respectively, are obtained by using a microprobe system to focus d isplaced pump and probe laser beams in a microfabricated flow channel and t o detect the scattering. The fingerprint modes of both species are very sim ilar and characteristic of an all-trans chromophore, Lumi exhibits a relati vely normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while M eta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bath orhodopsin, Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bat horhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D 2O buffer. This indicates that the Schiff base hydrogen bonding is dramatic ally weakened in the bathorhodopsin to Lumi transition. However, the C=N st retching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deute ration-induced downshift of 24 cm(-1), identical to that of the all-trans p rotonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.