Direct Electrochemistry of Cofactor Redox Sites in a Bacterial Photosynthetic Reaction Center Protein

Conversion of light into chemical energy in photosynthesis is initiated in the reaction center (RC) protein. In photosynthetic bacteria, a series of one-electron steps occur in RC after photoexcitation of the primary electron donor (P), a dimer of bacteriochlorophylls (BChl). Electron acceptors include a bacteriopheophytin (BPhe) and a quinone (Q sub(A)). Herein we show that redox centers P, BPhe, and Q sub(A) in an RC protein in layered films of phospholipid or polycations exchange electrons reversibly with an underlying electrode. Midpoint potentials measured by direct voltammetry are in excellent agreement with values from mediated optical redox titrations (Table 1). This ability to achieve direct electron exchange with electrodes without mediators provides for rapid monitoring of electrochemical properties of bound cofactors using only tiny amounts of protein. Direct voltammetry may be inhibited by protein surface denaturation, lack of electrical access to prosthetic groups, or unfavorable orientation of proteins at electrodes. Thus, while pigments, BChl and BPhe, isolated from the reaction center (RC) protein displayed reversible voltammograms in solution, to our knowledge, no direct voltammetry on the RC protein has been reported previously. Various strategies can achieve direct electron exchange between electrodes and water-soluble proteins. Studies of membrane proteins are rare, but voltammetry of cyt c oxidase in lipid films has been described. We recently developed two film types which facilitate direct voltammetry of proteins. In the first, a mixture of lipid vesicles and protein are deposited onto a surface, yielding films ordered in multiple lipid bilayers. In the second method, alternate layers of polyions and proteins are grown on a surface. Both methods provided films which gave reversible electron transfer between electrodes and iron heme proteins or ferredoxins, whereas only very slow electron transfer occurred for these proteins in solution with bare electrodes. In this work, we report direct reversible voltammetry for the photosynthetic RC from purple bacterium, Rhodobacter (Rb.) sphaeroides wild-type strain 2.4.1, incorporated into films of dimyristoylphosphatidylcholine (DMPC) on graphite or indium tin oxide electrodes, or sandwiched between polycation layers on gold electrodes. RCs from wild-type Rb. sphaeroides strain 2.4.1 were obtained with negligible amounts of quinone Q sub(B) as described previously.