A MUTANT RHODOPSIN PHOTOPRODUCT WITH A PROTONATED SCHIFF-BASE DISPLAYS AN ACTIVE-STATE CONFORMATION - A FOURIER-TRANSFORM INFRARED-SPECTROSCOPY STUDY

In the rhodopsin mutant E113A/A117E the position of the protonated Sch iff base counterion, Glu(113), is moved by one helix turn from positio n 113 to 117. The photoreaction of this mutant pigment was studied by Fourier-transform infrared (FTIR) difference spectroscopy. At acidic p H, formation of a 474-nm absorbing photoproduct previously characteriz ed biochemically as a species that activates transducin caused infrare d absorption changes typical of metarhodopsin II (MII) formation in na tive rhodopsin. Specific spectral alterations revealed a localized per turbation near the protonated Schiff base in the dark state. In additi on, an infrared band assigned to the C=O stretching vibration of Glu(1 13) in MII of rhodopsin was abolished in the mutant. Absorption change s caused by Asp(83) and Glu(122) C=O stretching vibrations characteris tic of rhodopsin MII formation were not affected. At alkaline pH, muta nt E113A/A117E formed predominantly a 382-nm absorbing photoproduct. I t displayed infrared-difference absorption bands significantly differe nt from those of native MII over a large spectral range. These results support the conclusion that the 474-nm photoproduct of mutant E113A/A 117E, despite a protonated Schiff base linkage, displays a predominant ly MII-like conformation capable of catalyzing guanine-nucleotide exch ange by transducin.