RECONSTITUTION OF BACTERIORHODOPSIN FROM THE APOPROTEIN AND RETINAL STUDIED BY FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-tran sform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occ upies the binding site but does not reconstitute the chromophore was u sed as ''caged retinal''. Photoisomerization to the all-trans isomer t riggers the reconstitution reaction. Absorption bands in the FTIR diff erence spectra of the educt and product of the reaction could be assig ned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman sp ectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl gro ups of D96 and D115 by use of the mutants D115N and D96N. Both asparti c acids are protonated also in the apoprotein with pK(a) values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indica tion was found for protonation changes of carboxyl groups or other pro tonatable residues when carrying out the reaction at pH values between 4 and 10. The pi-I-dependent protonation changes reported earlier [Fi scher & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be ca used by protons in a hydrogen-bonded network. Mutations of E204, but n ot of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, with out changing its protonation state itself, is part of a protonatable h ydrogen-bonded network which changes its pK(a) during reconstitution t hereby causing the observed protonation changes.