Mechanism of Proton Transfer Inhibition by Cd2+Binding to Bacterial Reaction Centers: Determination of the pKAof Functionally Important Histidine Residues

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Abstract
The bacterial photosynthetic reaction center (RC) uses light energy to catalyze the reduction of a bound quinone molecule QB to quinol QBH2. In RCs from Rhodobacter sphaeroides the protons involved in this process come from the cytoplasm and travel through pathways that involve His-H126 and His-H128 located near the proton entry point. In this study, we measured the pH dependence from 4.5 to 8.5 of the binding of the proton transfer inhibitor Cd2+, which ligates to these surface His in the RC and inhibits proton-coupled electron transfer. At pH 7, KD becomes essentially independent of pH. A theoretical fit to the data over the entire pH range required two protons with pKA values of 6.8 and 6.3 (±0.5). To assess the contribution of His-H126 and His-H128 to the observed pH dependence, KD was measured in mutant RCs that lack the imidazole group of His-H126 or His-H128 (His → Ala). In both mutant RCs, KD was approximately pH independent, showing that Cd2+ does not displace protons upon binding in the mutant RCs, in contrast to the native RC in which His-H126 and His-H128 are the predominant contributors to the observed pH dependence of KD. Thus, Cd2+ inhibits RC function by binding to functionally important histidines.