Light-driven water splitting by plants, algae and cyanobacteria is pivotal for global bioenergetics and biomass formation. A manganese cluster bound to the photosystem-II proteins catalyzes the complex reaction at high rate, but the rate-determining factors are insufficiently understood. Here we traced the oxygen-evolution transition by time-resolved polarography and infrared spectroscopy for cyanobacterial photosystems genetically modified at two strategic sites, complemented by computational chemistry. We conclude three rate-determining functions of the protein environment of the metal cluster: acceleration of proton-coupled electron transfer, acceleration of substrate-water insertion after O 2-formation, and balancing of rate-determining enthalpic and entropic contributions. Whereas in general the substrate-water insertion step may be unresolvable in time-resolved experiments, here it likely became traceable because of deceleration by genetic modification. Our results may stimulate new time-resolved experiments on substrate-water insertion in photosynthesis, clarification of enthalpy-entropy compensation in enzyme catalysis, and knowledge-guided development of inorganic catalyst materials.