title: Detection of the water-binding sites of the oxygen-evolving complex of Photosystem II using W-band 17O electron-electron double resonance-detected NMR spectroscopy.
abstract: Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) 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 (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca 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 (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) 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(4).
authors.affiliation: Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Leonid
authors.initials: L
authors.lastname: Rapatskiy
authors.forename: Nicholas
authors.initials: N
authors.lastname: Cox
authors.forename: Anton
authors.initials: A
authors.lastname: Savitsky
authors.forename: William M
authors.initials: WM
authors.lastname: Ames
authors.forename: Julia
authors.initials: J
authors.lastname: Sander
authors.forename: Marc M
authors.initials: MM
authors.lastname: Nowaczyk
authors.forename: Matthias
authors.initials: M
authors.lastname: Rögner
authors.forename: Alain
authors.initials: A
authors.lastname: Boussac
authors.forename: Frank
authors.initials: F
authors.lastname: Neese
authors.forename: Johannes
authors.initials: J
authors.lastname: Messinger
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lubitz
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D017895:Manganese Compounds / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000378:metabolism*; D019712:Thermococcus / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D017895:Manganese Compounds; D045332:Photosystem II Protein Complex; D014867:Water