Flash-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the water-oxidizing reactions in the oxygen-evolving centre of photosystem II. Reactions of water molecules were directly monitored by detecting the OH stretching bands of weakly H-bonded OH of water in the 3700–3500 cm
−1
region in FTIR difference spectra during S-state cycling. In the S
1
→S
2
transition, a band shift from 3588 to 3617 cm
−1
was observed, indicative of a weakened H-bond. Decoupling experiments using D
2
O : H
2
O (1 : 1) showed that this OH arose from a water molecule with an asymmetric H-bonding structure and this asymmetry became more significant upon S
2
formation. In the S
2
→S
3
, S
3
→S
0
and S
0
→S
1
transitions, negative bands were observed at 3634, 3621 and 3612 cm
−1
, respectively, representing formation of a strong H-bond or a proton release reaction. In addition, using complex spectral features in the carboxylate stretching region (1600–1300 cm
−1
) as ‘fingerprints’ of individual S-state transitions, pH dependency of the transition efficiencies and the effect of dehydration were examined to obtain the information of proton release and water insertion steps in the S-state cycle. Low-pH inhibition of the S
2
→S
3
, S
3
→S
0
and S
0
→S
1
transitions was consistent with a view that protons are released in the three transitions other than S
1
→S
2
, while relatively high susceptibility to dehydration in the S
2
→S
3
and S
3
→S
0
transitions suggested the insertion of substrate water into the system during these transitions. Thus, a possible mechanism of water oxidation to explain the FTIR data is proposed.
Water oxidation mechanism in photosystem II, including oxidations, proton release pathways, O―O bond formation and O2 release
