INTERACTION OF ASPARTATE-85 WITH A WATER MOLECULE AND THE PROTONATED SCHIFF-BASE IN THE L-INTERMEDIATE OF BACTERIORHODOPSIN - A FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDY

Fourier-transform infrared spectra were recorded at 170 K before and a fter irradiating the Asp(85)--> Asn mutant of bacteriorhodopsin. The d ifference spectrum exhibits protein bands such as those due to the per turbations of Asp(96) and Asp(115) and the N-H stretching vibration of tryptophan, characteristic of the L minus all-trans-bacteriorhodopsin spectrum of the wild-type protein. However, some vibrational bands of the peptide backbone and the chromophore are different from L and mor e characteristic of N of the wild-type protein. Remarkably, the shift observed for the vibrational band due to an internal water molecule up on L formation [Maeda, Sasaki, Shichida, and Yoshizawa (1992) Biochemi stry 31, 462-467] is absent. These changes in the spectrum of the muta nt could originate from the destruction of a hydrogen-bonding system c onsisting of Asp(85), the water molecule, and the Schiff base, upon re placement of Asp(85) with asparagine. These observations constitute di rect evidence for the interaction of water with Asp(85) at the time wh en it is protonated by the Schiff base.