Independent Mutation of Two Bridging Carboxylate Ligands Stabilizes Alternate Conformers of the Photosynthetic O₂-evolving Mn₄CaO₅ Cluster in Photosystem II

The O₂-evolving Mn₄CaO₅ cluster in photosystem II is ligated by six carboxylate residues. One of these is D170 of the D1 subunit. This carboxylate bridges between one Mn ion (Mn4) and the Ca ion. A second carboxylate ligand is D342 of the D1 subunit. This carboxylate bridges between two Mn ions (Mn1 and Mn2). D170 and D342 are located on opposite sides of the Mn₄CaO₅ cluster. Recently, it was shown that the D170E mutation perturbs both the intricate networks of H-bonds that surround the Mn₄CaO₅ cluster and the equilibrium between different conformers of the cluster in two of its lower oxidation states, S₁ and S₂, while still supporting O₂ evolution at approximately 50% the rate of the wild type. In this study, we show that the D342E mutation produces much the same alterations to the cluster’s FTIR and EPR spectra as D170E, while still supporting O₂ evolution at approximately 20% the rate of the wild type. Furthermore, the double mutation, D170E + D342E, behaves similarly to the two single mutations. We conclude that D342E alters the equilibrium between different conformers of the cluster in its S₁ and S₂ states in the same manner as D170E and perturbs the H-bond networks in a similar fashion. This is the second identification of a Mn₄CaO₅ metal ligand whose mutation influences the equilibrium between the different conformers of the S₁ and S₂ states without eliminating O₂ evolution. This finding has implications for our understanding of the mechanism of O₂ formation in terms of catalytically active/inactive conformations of the Mn₄CaO₅ cluster in its lower oxidation states.