Identification of Oxidized Amino Acid Residues in the Vicinity of the Mn4CaO5 Cluster of Photosystem II: Implications for the Identification of Oxygen Channels within the Photosystem

Photosystem II (PS II) functions as a light-driven, water-plastoquinone oxidoreductase. In higher plants and cyanobacteria at least six intrinsic proteins appear to be required for O2 evolution. These are CP47, CP43, D1, D2, and the α and β subunits of cytochrome b559. Deletion of these subunits uniformly results in the loss of PS II function and assembly (1, 2). Additionally, in higher plants, three extrinsic proteins, PsbO, PsbP and PsbQ are also required for maximal rates of O2 evolution under physiological inorganic cofactor concentrations (3). Of these three proteins, the PsbO protein appears to play a central role in the stabilization of the manganese cluster, is essential for efficient and stable O2 evolution and is required, along with PsbP, for photoautotrophic growth and PS II assembly in higher plants propagated under normal growth conditions (4–6). Under low light growth conditions the PsbQ component is also required for photoautotrophy (7). Over the past eleven years, moderate resolution crystal structures of cyanobacterial PS II have significantly enhanced our understanding of the molecular organization of the constituent polypeptides of the photosystem and the active site for oxygen evolution, the Mn4O5Ca cluster (8–12). Recently, a high resolution 1.9 A crystal structure of cyanobacterial PS II has been presented (13). It should be noted that no crystal structures for higher plant PS II are available. While there are differences between the higher plant and the cyanobacterial photosystems, particularly with respect to the identity and organization of the extrinsic proteins (3), the amino acid sequences of the intrinsic components (D1, D2, CP43 and CP47) are nearly identical (>85% similarity, (14)). Consequently, one would expect that the structural organization of these components within PS II would be highly homologous between higher plants and cyanobacteria. The production of molecular oxygen by PS II is accompanied by the unavoidable possibility of oxidative modification of amino acid residues within the PS II complex. While one would predict that amino acid residues located at the surface of the complex which are in contact with the bulk, oxygenated solvent would be susceptible to such modification, buried amino acid residues in the vicinity of the Mn4CaO5 cluster, the active site for water oxidation, might also be particularly susceptible to oxidative modification. This is exacerbated by the production of reactive oxygen species (ROS), such as H2O2, O2•− and OH• at, or in the vicinity of, the Mn4CaO5 cluster under a variety of stress conditions (15). Indeed, earlier studies have identified the double oxidation of the CP43 residue 365W to form an N-formylkynurenine derivative (16, 17). This residue is located 17 A from Mn2 of the Mn4CaO5 cluster. Given the possibility that oxidative modification of PS II could lead to damage of the photosystem, several groups have hypothesized that channels present in PS II would function to vector molecular oxygen and, by extension, ROS away from the Mn4CaO5 cluster towards the surface of the photosystem. Murray and Barber (18) used the CAVER Program (19) to examine the 3.5 A crystal structure of T. elongatus (10); later examining similar PS II crystals perfused with xenon (20). Gabdulkhakov et al. (21) used nobel gas and dimethyl sulfoxide co-crystallization studies in combination with CAVER to examine the 2.9 A structure of T. elongatus (12), and Ho and Styring calculated solvent-accessible surfaces for the 3.0 A T. elongatus structure of Loll et al. (11). We hypothesized that amino acid residues in contact with such oxygen channels in the interior of PS II would be significantly more susceptible to oxidative amino acid modification than residues not exposed to molecular oxygen and/or ROS. Consequently, the identification of such oxidatively modified residues in the interior of PS II should serve to complement and extend the largely computational studies mentioned above.