Electronic structure of the tyrosine D radical and the water-splitting complex from pulsed ENDOR spectroscopy on photosystem II single crystalsw Christian Teutloff, a Susanne Pudollek, a Sven Keßen, a Matthias Broser, b Athina Zouni b and Robert Bittl* a Received 22nd April 2009, Accepted 30th June 2009 First published as an Advance Article on the web 14th July 2009 DOI: 10.1039/b908093g Pulsed electron nuclear double resonance (ENDOR) spectroscopy at Q- and W-band frequencies was applied to single crystals of photosystem II from Th. elongatus. W-Band 1 H-ENDOR on the dark-stable radical state Y  D of the redox-active tyrosine residue Y D yields a complete mapping of the electronic structure of this amino acid radical in terms of an assignment of all hyperfine coupling tensors of the protons covalently bound to the side chain. This study can serve as a model case for the potential of high-field/high-frequency ENDOR on protein single crystals for obtaining highly resolved electronic structure information. Q-band 55 Mn-ENDOR was applied to the S 2 oxidation state of the water-splitting complex in photosystem II single crystals. Irrespective of the difficulties arising from the extremely broad electron paramagnetic resonance (EPR) spectroscopy (E200 mT) and ENDOR (E100 MHz) spectra a tentative assignment of the Mn ion in the formal oxidation state III to a Mn position in the structural model of PSII is possible on the basis of the ENDOR data. Introduction The two large membrane-bound pigment–protein complexes photosystem I (PSI) and photosystem II (PSII) are the sites of the light reactions in oxygenic photosynthesis. PSI is a light- driven plastocyanin : ferredoxin oxidoreductase transferring electrons from plastocyanin on the lumenal side to ferredoxin on the stromal side of the thylakoid membrane, in essence working as a molecular solar cell. 1 PSII is a light-driven water : plastoquinone oxidoreductase transferring electrons from water on the lumenal side to an exchangeable plasto- quinone cofactor at the stromal side. 2 Molecular oxygen is formed as a by-product of the light-driven water oxidation in PSII. The catalytic site for the water splitting and oxygen evolution, the water-oxidising complex (WOC), is a manganese– calcium complex consisting of four manganese ions and a calcium ion connected via several m-oxo bridges (Mn 4 O x –Ca complex). 3,4 The WOC is coupled to the chlorophyll moieties making up the site of initial electron transfer after photo- excitation by a redox-active tyrosine amino acid residue (Y Z ). 5,6 The cofactors of the electron transfer chain in PSII are embedded in the hetero-dimeric reaction centre core of two protein subunits, D1 and D2. These two subunits show a high sequence homology and are arranged in a pseudo-C 2 symmetry leading to two almost symmetric branches of electron transfer cofactors except the WOC. Symmetrically to Y Z , a second redox-active tyrosine residue, Y D , is found. This residue is also coupled to the electron transfer chain and forms under light illumination a dark-stable radical, Y  D . The func- tional role of Y D is so far not understood in detail and mutants lacking Y D are able to maintain photo-autotrophic growth. 7 Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool in the study of photosynthesis as the light-induced single-electron transfer leads to a sequence of paramagnetic intermediates of the organic electron transfer cofactors 8 as well as the WOC. 9 The four-electron reaction of water oxidation requires the accumulation of four oxidising equivalents in the WOC. In the model for water oxidation put forward by Kok et al., 10 the WOC passes through four (meta)stable oxidation states S 0–3 and a transition state S 4 . The dark-stable ground state is the S 1 -state. The S 2 -state generated after one photon absorption is paramagnetic and has extensively been studied by EPR techniques. 11–16 The initial observation of a multi-line signal (MLS) in EPR spectroscopy by Dismukes and Siderer 17 was the first evidence for the involvement of a mixed-valent manganese cluster in the WOC. Even after the availability of structural models for PSII from X-ray crystallography 18–23 information on the geometric and electronic structure of the WOC from spectroscopy remains essential. Owing to radiation damage 24 the current X-ray structures of the WOC probably do not correspond to any of the functional S-state structures. Furthermore, neither the bridging m-oxo nor the amino acid ligands have been assigned unambiguously by X-ray crystallography so far. An a Fachbereich Physik, Freie Universita ¨t Berlin, Berlin, Germany. E-mail: robert.bittl@fu-berlin.de b Max-Volmer-Laboratorium, Technische Universita ¨t Berlin, Berlin, Germany w Electronic supplementary information (ESI) available: Orientational information of the single crystals used for Y  D 1 H-ENDOR at W-band and WOC S 2 -state 55 Mn-ENDOR at Q-band in the labora- tory frames, complete 94 GHz single crystal Y  D ENDOR data sets (including simulations of the individual spectra), and Q-band Y  D EPR for the single crystal used for the WOC S 2 -state 55 Mn-ENDOR are given. See DOI: 10.1039/b908093g This journal is  c the Owner Societies 2009 Phys. Chem. Chem. Phys., 2009, 11, 6715–6726 | 6715 PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics