Application of near-IR Fourier transform resonance Raman spectroscopy to the study of photosynthetic proteins

Abstract

We demonstrate the application of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy using 1064 nm excitation to obtain high quality preresonance Raman spectra of bacteriochlorophyll chromophores in photosynthetic proteins from purple bacteria at room temperature without sample degradation. We present NIR FT preresonance Raman spectra of chromatophores from Rhodospirillum (Rsp.) rubrum, carotenoidless strain G9 containing bacteriochlorophyll a (BChl a) chromophores mostly from its B880 antenna complex, and the B850-800 antenna complex from Rhodobacter (Rb.) sphaeroides, 2.4.1 strain; these spectra are compared with their resonance Raman (RR) spectra obtained using 363.8 nm excitation at 30 K. For antenna complexes not containing carotenoid the FT Raman spectra are dominated by the vibrational modes of the bacteriochlorophyll chromophores with no interference from the modes of the protein or membrane. In the NIR FT Raman spectrum of the B850-800 complex from Rb. sphaeroides 2.4.1, strong contributions from the carotenoid molecule are observed to cause some interference with the 1609 cm −1 band of the BChl a molecules. We also present FT Raman spectra of reaction centers (RCs) from Rb. sphaeroides R 26 in the reduced and oxidized states of their primary electron donor (P). In the reduced state, it is estimated that ca 70% of the FT Raman spectrum arises from reduced P whose electronic absorption band is at 865 nm. With 1064 nm excitation of the RCs poised in the oxidized cation radical state of P, we have observed for the first time a Raman spectrum of P +. via resonance in the vibronic region of the 1250 nm absorption band of this species. This spectrum indicates that the unpaired electron in dimeric P +. is not equally shared between the two BChl molecules constituting the primary donor. Spectral contributions of the carotenoid in the FT RR spectra of wild-type Rb. sphaeroides (2.4.1) RCs confirm it is assuming an out-of-plane distorted, 15- cis configuration under the specific conditions offered by FT Raman spectroscopy, i.e. with the sample at room temperature and using an excitation wavelength which is non-actinic for the carotenoid.