The site of biological water oxidation is highly conserved across photosynthetic organisms, but differences of unidentified structural and electronic origin exist between taxonomically discrete clades, revealed by distinct spectroscopic signatures of the oxygen-evolving Mn4CaO5 cluster and variations in active-site accessibility. Comparison of atomistic models of a native cyanobacterial form (Thermosynechococcus vulcanus) and a chimeric spinach-like form of photosystem II allows us to identify the precise atomic-level differences between organisms in the vicinity of the manganese cluster. Substitution of cyanobacterial D1-Asn87 by higher-plant D1-Ala87 is the principal discriminating feature: it drastically rearranges a network of proximal hydrogen bonds, modifying the local architecture of a water channel and the interaction of second coordination shell residues with the manganese cluster. The two variants explain species-dependent differences in spectroscopic properties and in the interaction of substrate analogues with the oxygen-evolving complex, enabling assignment of a substrate delivery channel to the active site.