Perfusion- and Light-induced ATR-FTIR Redox Studies of Rb. Sphaeroides Photosynthetic Reaction Centers

Abstract

By combining attenuated total reflection (ATR) Fourier transform infrared (FTIR) difference spectroscopy with perfusion of chemical compounds on membrane proteins deposited on the surface of an internal reflection prism, we demonstrate that redox transitions in various bioenergetic systems can be probed. After partial drying of reaction centers (RCs) from Rb. sphaeroides on the ATR prism, the film was rehydrated with buffer. The IR beam penetrates the ATR prism and the evanescent wave propagates through the sample film which is enclosed in a flow cell. The rRedox state changess in the RCs film were manipulated by changing the chemical composition (ferricyanide, ferrocyanide or dithionite) of the flowing buffer. Illumination of the same film was also performed to generate the P+QA- charge-separated state. Chemically-induced ATR/FTIR difference spectra were recorded ofcorresponding to (a) the oxidation of the primary donor (P+/P); and to (b) the reduction of the primary quinone acceptor (QA-/QA); were obtained, as well as ; as well as (c) the light-induced P+QA-/PQA difference spectrum. The calculated QA-/QA spectrum (c-a) agrees with the chemically-induced spectrum (b). ATR/FTIR spectroscopic studies on RCs from Rps. viridis wand PS I will be also presented. More generally, the perfusion-induced ATR/FTIR technique can be widely applied to investigate molecular vibrational changes triggered by redox- and pH-changes, or ligand binding.