| Sumario: | Water binding to the MnOCa cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S state was studied via HO- and HO-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating O (I = /) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal N (I = /) labeling was employed to clearly discriminate the O hyperfine couplings that overlap with N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH ligand; and (iii) Mn/Ca-HO ligand(s). These assignments are based on O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH perturbation of the O signal envelope and density functional theory calculations. The relative orientation of the putative O μ-oxo bridge hyperfine tensor to the N(N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the MnOCa open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All O signals were seen even after very short (≤15 s) incubations in HO suggesting that all exchange sites identified could represent bound substrate in the S state including the μ-oxo bridge. H/H (I = /, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small H/H couplings observed require that all the μ-oxo bridges of the MnOCa cluster are deprotonated in the S state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S.
|
|---|