Proton release due to manganese binding and oxidation in modified bacterial reaction centers

The pH dependence of binding and oxidation of Mn²⁺ in highly oxidizing reaction centers with designed metal-binding sites was characterized by light-minus-dark optical difference spectroscopy and direct measurements of proton uptake/release. These mutants bind a Mn²⁺ ion that can efficiently transfer an electron to the oxidized bacteriochlorophyll dimer, as described earlier [Thielges et al. (2005) Biochemistry 44, 7389-7394]. The dissociation constant, K_D, significantly increased with decreasing pH. The pH dependence of K_D between pH 7 and pH 8 was consistent with the binding of Mn²⁺ being stabilized by the electrostatic release of two protons. The strong pH dependence of proton release upon Mn²⁺ binding, with a maximal release of 1.4 H⁺ per reaction center, was interpreted as being a result of a shift in the pK_a values of the coordinating residues and possibly other nearby residues. A small amount of proton release associated with Mn²⁺ oxidation was observed upon illumination. These results show that functional metal-binding sites can be incorporated into proteins upon consideration of both the metal coordination and protonation states of the ligands.