Abstract
Rapid-scan FTIR difference spectroscopy was used to investigate light-induced reduction of the ubiquinone QB, and the oxidation kinetics of QB–and QH2by external mediators in isolated reaction centers (RCs) fromR. 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 TMPDredrapidly 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 TMPDoxoxidises both QB–and QH2faster than DADox. In particular, QB–is oxidised faster than QH2at all temperatures studied.Results are discussed in the framework of time-resolved infrared studies onR. sphaeroidesRCs, showing advantages/drawbacks of the proposed experimental approach.
Highlights
The photosynthetic reaction center (RC) from R. sphaeroides is one of the most-studied membrane enzymes [16,17,28,36].The structure of the R. sphaeroides reaction centers (RCs) has been determined with up to 1.87 Å resolution [18] leading to a detailed knowledge of the position of pigments, cofactors, amino acids, as well as on their relative orientation and distance
The ubiquinol QBH2 formed leaves the RC to diffuse in the membrane and is replaced by a new ubiquinone coming from the membrane
In these experiments ferrocyanide and TMPD were used as external electron donors to reduce P870+ [20,21]
Summary
The photosynthetic reaction center (RC) from R. sphaeroides is one of the most-studied membrane enzymes [16,17,28,36].The structure of the R. sphaeroides RC has been determined with up to 1.87 Å resolution [18] leading to a detailed knowledge of the position of pigments, cofactors, amino acids, as well as on their relative orientation and distance. The photosynthetic reaction center (RC) from R. sphaeroides is one of the most-studied membrane enzymes [16,17,28,36]. Protons are transferred towards the QB site through a pathway formed by protonable amino acid side chains and water molecules [11,28,29,36]. The ubiquinol QBH2 formed leaves the RC to diffuse in the membrane and is replaced by a new ubiquinone coming from the membrane. The ubiquinol is reoxidised by the cytochrome bc complex (another enzyme present in the membrane); the overall effect of this series of reactions is to move H+ from the cytoplasm to the periplasm.
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