Flash-induced FTIR difference spectroscopy shows no evidence for the structural coupling of bicarbonate to the oxygen-evolving Mn cluster in photosystem II

Bicarbonate is known to be required for the maximum activity of photosystem II. Although it is well established that bicarbonate is bound to the nonheme iron to regulate the quinone reactions, the effect of bicarbonate on oxygen evolution is still controversial, and its binding site and exact physiological roles remain to be clarified. In this study, the structural coupling of bicarbonate to the oxygen-evolving center (OEC) was studied using Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra during the S-state cycle of OEC were recorded using the PSII core complexes from Thermosynechococcus elongatus in the presence of either unlabeled bicarbonate or (13)C-bicarbonate. The H (12)CO 3 (-)-minus-H (13)CO 3 (-) double difference spectra showed prominent bicarbonate bands at the first flash, whereas no appreciable bands were detected at the second to fourth flashes. The bicarbonate bands at the first flash were virtually identical to those from the nonheme iron, which was preoxidized by ferricyanide and photoreduced by a single flash, recorded using Mn-depleted PSII complexes. Using the bicarbonate bands of the nonheme iron as an internal standard, it was concluded that no bicarbonate band arising from OEC exists in the S-state FTIR spectra. This conclusion indicates that bicarbonate is not affected by the structural changes in OEC upon the four S-state transitions. It is thus strongly suggested that bicarbonate is neither a ligand to the Mn cluster nor a cofactor closely coupled to OEC, although the possibility cannot be fully excluded that nonexchangeable bicarbonate exists in OEC as a constituent of the Mn-cluster core. The data also provide strong evidence that bicarbonate does not function as a substrate or a catalytic intermediate. Bicarbonate may play major roles in the photoassembly process of the Mn cluster and in the stabilization of OEC by a rather indirect interaction.