Monitoring Water Reactions during the S-State Cycle of the Photosynthetic Water-Oxidizing Center: Detection of the DOD Bending Vibrations by Means of Fourier Transform Infrared Spectroscopy

Abstract

Photosynthetic water oxidation takes place in the water-oxidizing center (WOC) of photosystem II (PSII). To clarify the mechanism of water oxidation, detecting water molecules in the WOC and monitoring their reactions at the molecular level are essential. In this study, we have for the first time detected the DOD bending vibrations of functional D 2O molecules during the S-state cycle of the WOC by means of Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra upon S-state transitions were measured using the PSII core complexes from Thermosynechococcus elongatus moderately deuterated with D 2 (16)O and D 2 (18)O. D 2 (16)O-minus-D 2 (18)O double difference spectra at individual S-state transitions exhibited six to eight peaks arising from the D (16)OD/D (18)OD bending vibrations in the 1250-1150 cm (-1) region. This observation indicates that at least two water molecules, not in any deprotonated forms, participate in the reaction at each S-state transition throughout the cycle. Most of the peaks exhibited clear counter peaks with opposite signs at different transitions, reflecting a series of reactions of water molecules at the catalytic site. In contrast, negative bands at approximately 1240 cm (-1) in the S 2 --> S 3, S 3 --> S 0, and possibly S 0 --> S 1 transitions, for which no clear counter peaks were found in other transitions, can be interpreted as insertion of substrate water into the WOC from a water cluster in the proteins. The characteristics of the weakly D-bonded OD stretching bands were consistent with the insertion of substrate from internal water molecules in the S 2 --> S 3 and S 3 --> S 0 transitions. The results of this study show that FTIR detection of the DOD bending vibrations is a powerful method for investigating the molecular mechanism of photosynthetic water oxidation as well as other enzymatic reactions involving functional water molecules.

Substrate–water exchange in photosystem II is arrested before dioxygen formation

“…Mn III/IV , purple; Ca 2+ , yellow; oxo-bridges, red; water–oxygens, blue; Cl − , green; amino acid backbones, grey; carboxy-oxygen, red; and His-nitrogen, dark blue. ( b ) Kok cycle for water oxidation in PSII including proton release to the bulk822 (see also Supplementary Note 1) and water-binding events2728. The S i states ( i =0, …, 4) denote the oxidation state of the Mn 4 CaO 5 cluster relative to the S 0 state, while the plus sign indicates a positive extra charge.…”

“…It is widely agreed that this dioxygen-forming reaction sequence starts with a proton release during the → transition22232425, followed by the formation of a Ca- or Mn-bound oxyl radical or of a Mn V -oxo group due to oxidation of the cluster by 61426. Although the following reaction steps towards O 2 must include the formation of a bound peroxidic intermediate, dioxygen formation and release, and the rebinding of substrate water2728 (Fig. 1c), the exact nature of the chemistry involved remains unsettled, as these transient states have so far largely eluded biophysical investigations6142829303132.…”

QM/MM study of the S2 to S3 transition reaction in the oxygen-evolving complex of photosystem II

“…On the other hand, for the S1 → S2 transition, the OEC core geometry was almost unchanged, and no proton release was observed. Thus, the S1 → S2 transition is expected to be a simple one-electron oxidation reaction [4,8]. Therefore, the first chemical reaction of PSII-OEC is considered to occur during the S2 → S3 transition.…”

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…[32][33][34] Changes in the D-O-D bending of water molecules have been detected, as well. 35 It was shown that the exchange of the protein solvent from H 2 16 O to H 2 18 O is enough to detect dangling waters in the dark state of BR, without the need to induce a photoreaction. 36 In the present work, another class of the large family of retinylidene proteins was studied.…”