Time-Resolved Step-Scan FT-IR Investigations of the Transition from KL to L in the Bacteriorhodopsin Photocycle: Identification of Chromophore Twists by Assigning Hydrogen-Out-Of-Plane (HOOP) Bending Vibrations

Abstract

Sub-microsecond time-resolved step-scan FT-IR spectroscopy is applied to the study of the molecular changes and their dynamics occurring during the KL-L transition of bacteriorhodopsin. The time-resolved difference spectra are compared to the static low-temperature BR → K and BR → L difference spectra. Our data show that the protein part in KL is similar to that in K. However, the chromophore is more relaxed and is differently twisted. A strong hydrogen-out-of-plane (HOOP) mode in KL is assigned to the 15-HOOP. As is the case for L, a strong deformation of the C14-C15single bond is deduced for KL. Evidence of a KL → L equilibrium is presented. In N, a 15-HOOP mode similar to that in L is observed, indicating very similar twists of the C14-C15single bond. This observation excludes major contributions of this deformation to the reduction of the pKaof the Schiff base in L. From the spectral changes, important molecular events are deduced that occur in the transitions to KL, L, and N.