Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic
photosynthesis, the process that powers life on Earth, and is the paradigm for
engineering solar fuel–production systems. Each complete reaction cycle of
photosynthetic water oxidation requires the removal of four electrons and four
protons from the catalytic site, a manganese–calcium complex and its protein
environment in photosystem II. In time-resolved photothermal beam deflection
experiments, we monitored apparent volume changes of the photosystem II
protein associated with charge creation by light-induced electron transfer
(contraction) and charge-compensating proton relocation (expansion). Two
previously invisible proton removal steps were detected, thereby filling two
gaps in the basic reaction-cycle model of photosynthetic water oxidation. In
the S2 → S3 transition of the classical S-state cycle, an intermediate is
formed by deprotonation clearly before electron transfer to the oxidant
(Graphic). The rate-determining elementary step (τ, approximately 30 µs at 20
°C) in the long-distance proton relocation toward the protein–water interface
is characterized by a high activation energy (Ea = 0.46 ± 0.05 eV) and strong
H/D kinetic isotope effect (approximately 6). The characteristics of a proton
transfer step during the S0 → S1 transition are similar (τ, approximately 100
µs; Ea = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however,
the proton removal from the Mn complex proceeds after electron transfer to
Graphic. By discovery of the transient formation of two further intermediate
states in the reaction cycle of photosynthetic water oxidation, a temporal
sequence of strictly alternating removal of electrons and protons from the
catalytic site is established
