Ammonia Binding to the Oxygen-Evolving Complex Probed by High-Energy Resolution Fluorescence Detected X-Ray Absorption Spectroscopy

The insertion pathways and binding sites of substrate water molecules at the catalytic Mn₄CaO₅ cluster of the oxygen-evolving complex (OEC) in photosystem II (PSII) remain a fundamentally unresolved question toward understanding biological water oxidation. To address this question, small molecules have been employed as "water analogues" to probe substrate binding to the OEC. In this context, the binding of ammonia has been extensively investigated and discussed using spectroscopic, structural, and quantum chemical methods, but a definitive answer regarding the ammonia binding site has not yet been achieved. Herein, we present high-energy resolution fluorescence detected (HERFD) Mn K-edge X-ray absorption spectroscopy (XAS) in ammonia-treated S₂ state samples of the OEC. Pre-edge features were correlated with possible structural models with the aid of quantum chemical calculations. The comparison of calculated and experimental difference spectra between the native and ammonia-treated samples allows us to evaluate different modes of ammonia interaction with the OEC. The combined spectroscopic and theoretical investigation suggests the substitution of the terminal water ligand W2 on Mn4 as the most plausible ammonia binding mode, followed closely by the substitution of the second terminal water ligand (W1), and the coordination of ammonia on Mn1 as a sixth ligand. Our results are in line with the leading interpretations of other spectroscopic and kinetic studies, converging on the conclusion that the Mn4 ion is either the most accessible or the strongest binding site for substrate analogues.