Vibrational Spectrum of K-590 Containing¹³C_14,15Retinal:  Picosecond Time-Resolved Coherent Anti-Stokes Raman Spectroscopy of the Room Temperature Bacteriorhodopsin Photocycle

Abstract

The C-C stretching vibrations (1100-1400 cm -1 ) of the K-590 intermediate (containing a 13 C 14,15 retinal), formed during the room-temperature (RT) bacteriorhodopsin (BR) photocycle, are measured using picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS). Although time-resolved resonance Raman data have been published previously for intermediates in the room-temperature BR photocycle, these PTR/ CARS data are the first time-resolved vibrational spectra from a picosecond BR intermediate at RT containing an isotopically-labeled ( 13 C) retinal. The C 14 and C 15 positions are selected for isotopic labeling because motions around the C 13 dC 14 and C 14 -C 15 bonds are thought to underlie the structural transformation from BR-570 to K-590 and, therefore, the energy storage and transduction mechanism in the RT/BR photocycle. These PTR/CARS data are recorded 50 ps after the BR photocycle is initiated with 570-nm (5 ps, fwhm) excitation and are fit to within <1 cm -1 via third-order nonlinear susceptibility (χ (3) ) relationships. Comparisons of these PTR/CARS data at RT with the results from earlier resonance Raman (RR) studies of K-590 at low temperature (LT) reveal new temperature effects. Specifically, three CARS bands (1197, 1184, and 1167 cm -1 ) are observed from 13 C 14,15 K-590 in H 2 O samples via PTR/CARS at RT, while only two bands (1189 and 1170 cm -1 ) are found in LT/RR measurements from 13 C 14,15 K-625. Analogous temperature-dependent differences are found in data measured from 13 C 14,15 K-590 in D 2 O samples. Independently, PTR/CARS data at RT demonstrate that deuteration of the Schiff-base nitrogen causes major changes in the fingerprint region: the 1197-cm -1 band decreases to 1193 cm -1 while diminishing in intensity by half and a new band appears at 1206 cm -1 . No such deuterium effect is observed in the LT/RR data from 13 C 14,15 K-590. The previously unrecognized sensitivity of fingerprint bands to deuteration of the Schiff-base nitrogen suggests that the C-C stretching modes are highly mixed with each other and coupled to the N-H(D) retinal rocking mode. Although the temperature and Schiff-base deuteration effects reported here had not been previously identified in LT vibrational data from K-625, an analysis of the RT/CARS data continues to support the 13-cis, 14-trans retinal structure in K-590 proposed from LT results.

Nanosecond retinal structure changes in K-590 during the room-temperature bacteriorhodopsin photocycle: picosecond time-resolved coherent anti-stokes Raman spectroscopy

“…The signal-to-noise (S/N) ratio found in time-resolved RR and IR data reported to date has not been sufficiently high to quantitatively compare vibrational specand Atkinson, 1989; Doig et al, 1991; Diller et al, 1991, delay times in the 10-ps to 260-ns time regime. The PTR/ CARS data reported here provide a unique opportunity to obtain such high S/N vibrational data as evidenced from recently published data from intermediates in the BR and rhodopsin photoreactions at RT (Ujj et al, 1996;Jager et al, 1996;Popp et al, 1996). Highly reproducible (<3% error functions) PTR/CARS spectra are obtained with accumulation times of <300 s. It is also important to note that excitation of the RT/BR photocycle in PTR/CARS experiments is achieved with a <3-ps pulse, which minimizes any secondary photochemistry and/or back-reactions that can significantly confuse the chemical reaction being investigated.…”

“…In all nine cases, these differences are significantly above the measurement noise as determined by repetitive experiments. Based on previous vibrational mode assignments of these bands in both time-resolved (Ujj et al, 1996;Jager et al, 1996;Weidlich and Siebert, 1993) and LT vibrational (Smith, 1985;Maeda et al, 1991) spectra, it is evident that the retinal structure near the C4-CI==N bonds (Schiff-base region) and along its polyene chain is altered during the 50-ps to 260-ns interval. These spectral changes may also reflect alterations in the protein adjacent to the Schiff base linkage.…”

“…A detailed description of the experimental setup and of the theoretical background for the PTRICARS measurements is described elsewhere (Ujj et al, 1994a(Ujj et al, ,b, 1996Jager et al, 1996). Only the actual measuring conditions used and the basics of the data analysis are described here.…”

“…The 1196-cm-1 band is the strongest in the fingerprint region (compared to the bands at 1188 cm-', 1206 cm-i, and 1220 cm-l). These bands are all assigned to highly mixed vibrations of C-C stretching modes (Jager et al, 1996). The 1196-cm-l band contains significant C,4-C,5 stretching character (Jager et al, 1996), as well as some hydrogen-in-plane rocking character (Smith, 1985).…”

“…Time-resolved vibrational spectra assignable to K-590 at RT have been measured with picosecond time resolution (Hsieh et al, 1981(Hsieh et al, , 1983; Stern and Mathies, 1985;Brack 1992;Atkinson and Ujj, 1992;Lohrmann et al, 1995;Althaus et al, 1995;Ujj et al, 1996). Recently, picosecond time-resolved coherent anti-Stokes Raman spectra (PTR/ CARS) from both native K-590 at RT and an isotopically labeled (I3C14,15) K-590 at RT have been reported Jager et al, 1996).…”