Time-Resolved Infrared Evidence for Protein Conformational Changes During the Dark-Rearrangement Process of Photosystem II Photoactivation: A Comparative Study of Solution and Crystal Samples

The catalytic site of photosynthetic water oxidation, the water-oxidizing complex (WOC), which contains the Mn₄CaO₅ cluster as its inorganic core, is assembled in photosystem II (PSII) through a light-driven process known as photoactivation. Despite extensive study, the detailed molecular mechanism underlying photoactivation remains elusive. Here, we investigated the mechanism of photoactivation by focusing on the "dark rearrangement process" that occurs following the first flash illumination, using time-resolved Fourier transform infrared (FTIR) measurements of apo-WOC PSII both in crystals, where the protein conformation remains nearly unchanged upon Mn depletion, and in solution, where Mn removal induces substantial conformational changes. Time-resolved FTIR spectra of apo-WOC PSII in solution, following single-flash illumination in the presence of Mn²⁺, revealed two distinct decay phases. The fast phase was characterized by increased relative intensities of amide I bands accompanied by shifts in carboxylate stretching bands, while the slow phase exhibited minimal spectral changes. In contrast, FTIR spectra of apo-WOC PSII in crystals showed only a single slow decay phase, with a time constant comparable to that of the slow component in solution, and with negligible change in spectral shape. This striking contrast between PSII in solution and in crystals provides definitive evidence that significant protein conformational changes, accompanied by Mn³⁺ relocation via carboxylate groups, occur during the dark rearrangement process following the initial photooxidation of Mn²⁺ under physiological conditions.