Electrochemical redox titration of cofactors in the reaction center fromRhodobacter sphaeroides

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Abstract
The electrochemical redox poising of the primary electron donor P and of the quinone electron acceptor(s) Q in isolated reaction centers from Rhodobacter sphaeroides in an ultra-thin-layer electrochemical cell, monitored by chronoamperometry and by spectroscopy in the visible/near-infrared region, is reported. Electrical application of a redox potential of +0.4 V (vs. Ag/AgCl/3 M KCl) leads to quantitative formation of the π-cation radical of P within a few minutes. The oxidized product can be re-reduced to the neutral species by application of 0 V, and full reversibility is maintained over many-cycles. By poising at a series of intermediate potentials, a titration curve for the 865 nm P band was obtained, which could be fitted to a Nernst function with E m = 0.485 vs. SHE and n = 0.96. By Application of negative potentials (−0.2 V and −0.45 V vs. Ag/AgCl/3 M KCl), the quinone electron acceptors were reversibly reduced as demonstrated by the shift of bacteriopheophytin absorption and drastically changed kinetics of charge recombination. The use of this thin-layer electrochemical technique for the determination of midpoint potentials, for the investigation of redox-poised electron transfer reactions as well as for spectroscopy in the mid-infrared region is discussed.