Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) of Light-Activated Photosynthetic Reaction Centers from Rhodobacter sphaeroides Reconstituted in a Biomimetic Membrane System

Abstract

Surface-enhanced IR absorption spectroscopy (SEIRAS) in the ATR configuration has been performed on reaction centers (RCs) from R. sphaeroides. Surface-enhancement is achieved by a thin, structured gold film present on the surface of an ATR crystal. Purified RCs are immobilized as a monolayer on top of the gold film via a poly his-tag engineered to the C-terminal end of the M subunit. Subsequently, the RCs are reconstituted into a lipid bilayer by in situ dialysis. Light-minus-dark absorbance spectra were recorded under continuous illumination using the spectrum in the dark as the reference. A number of strong bands have been observed indicating the excitation of the special pair as well as alterations of quinone/quinol species. Spectra were recorded at different time intervals with and without liposoluble Q10 coreconstituted into the lipid phase. A steady (photostationary) state was approached slowly and bands were found to increase or decrease reversibly on illumination and relaxation. Tentative assignments were made for some bands, based on previous FTIR measurements. The long time scale of these processes was tentatively explained in terms of interprotein reactions of RC molecules.