Rapid-scan FTIR difference spectroscopy applied to ubiquinone reduction in photosynthetic reaction centers: Role of redox mediators

Abstract

Rapid-scan FTIR difference spectroscopy was used to investigate light-induced reduction of the ubiquinone Q_B, and the oxidation kinetics of Q_B^– and QH₂ by external mediators in isolated reaction centers (RCs) from R. sphaeroides. As redox mediators, a Ferrocyanide/N,N,Nʹ,Nʹ-tetramethyl-p-phenylenediamine (TMPD) mixture and an Ascorbate/2,3,5,6-tetramethyl-p-phenylenediamine (DAD) mixture are compared.Results show that TMPD^red rapidly reduces the photoproduced P₈₇₀⁺ primary donor. The process is fast enough to record rapid-scan FTIR spectra devoid of P₈₇₀⁺ bands down to 260 K. Results show also that TMPD^ox oxidises both Q_B^– and QH₂ faster than DAD^ox. In particular, Q_B^– is oxidised faster than QH₂ at all temperatures studied.Results are discussed in the framework of time-resolved infrared studies on R. sphaeroides RCs, showing advantages/drawbacks of the proposed experimental approach.