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-C₁₅ single 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-C₁₅ single bond. This observation excludes major contributions of this deformation to the reduction of the pK_a of the Schiff base in L. From the spectral changes, important molecular events are deduced that occur in the transitions to KL, L, and N.