Bernhard Loll

The oxidation of water to dioxygen is catalyzed within photosystem II (PSII) by a Mn(4)Ca cluster, the structure of which remains elusive. Polarized extended x-ray absorption fine structure (EXAFS) measurements on PSII single crystals constrain the Mn(4)Ca cluster geometry to a set of three similar high-resolution structures. Combining polarized EXAFS and x-ray diffraction data, the cluster was placed within PSII, taking into account the overall trend of the electron density of the metal site and the putative ligands. The structure of the cluster from the present study is unlike either the 3.0 or 3.5 angstrom-resolution x-ray structures or other previously proposed models.

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn(4)Ca active site in single crystals of photosystem II as a function of dose and energy of x-rays, temperature, and time. These studies reveal that the conditions used for structure determination by x-ray crystallography cause serious damage specifically to the metal-site structure. The x-ray absorption spectra show that the structure changes from one that is characteristic of a high-valent Mn(4)(III(2),IV(2)) oxo-bridged Mn(4)Ca cluster to that of Mn(II) in aqueous solution. This damage to the metal site occurs at a dose that is more than one order of magnitude lower than the dose that results in loss of diffractivity and is commonly considered safe for protein crystallography. These results establish quantitative x-ray dose parameters that are applicable to redox-active metalloproteins. This case study shows that a careful evaluation of the structural intactness of the active site(s) by spectroscopic techniques can validate structures derived from crystallography and that it can be a valuable complementary method before structure-function correlations of metalloproteins can be made on the basis of high-resolution x-ray crystal structures.

In the crystal structure of photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus at 3.2 Å resolution, several loop regions of the principal protein subunits are now defined that were not interpretable previously at 3.8 Å resolution. The head groups and side chains of the organic cofactors of the electron transfer chain and of antenna chlorophyll a (Chl a) have been modeled, coordinating and hydrogen bonding amino acids identified and the nature of the binding pockets derived. The orientations of these cofactors resemble those of the reaction center from anoxygenic purple bacteria, but differences in hydrogen bonding and protein environment modulate their properties and provide the unique high redox potential (1.17 V) of the primary donor. Coordinating amino acids of manganese cluster, redox-active TyrZ and non-haem Fe2+ have been determined, and an all-trans β-carotene connects cytochrome b-559, ChlZ and primary electron donor (coordinates are available under PDB-code 1W5C).