X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn4Ca 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 Mn4(III2,IV2) oxo-bridged Mn4Ca 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.manganese ͉ oxygen evolution ͉ water oxidation ͉ x-ray spectroscopy

In the crystal structure of photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus at 3.2 A ˚resolution, several loop regions of the principal protein subunits are now defined that were not interpretable previously at 3.8 A ˚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 Tyr Z and non-haem Fe 21 have been determined, and an all-trans b-carotene connects cytochrome b-559, Chl Z and primary electron donor (coordinates are available under PDB-code 1W5C).

Nonphotochemical hole burning and pressure-dependent absorption and hole-burning results are presented for the isolated (disaggregated) chlorophyll a/b light-harvesting II trimer antenna complex of green plants. Analysis of the 4.2 K burn-fluence dependent hole spectra and zero-phonon hole action spectra indicates that the three lowest energy states (Q y ) lie at 677.1, 678.4 and 679.8 nm. Their combined absorption intensity is equivalent to that of three Chl a molecules. The inhomogeneous broadening of their absorption bands is 70 cm-1. It is argued that these states, separated by 30 cm-1, are associated with the lowest energy state of the trimer subunit with the 30 cm-1 separations due to the indigenous structural heterogeneity of protein complexes. The linear electron−phonon coupling of the 679.8 nm state is weak and characterized, in part, by a mean phonon frequency of ωm = 18 cm-1 and Huang−Rhys factor of S m = 0.8, values which yield the correct Stokes shift for fluorescence from the 679.8 nm state at 4.2 K. The temperature dependence of the zero-phonon hole (ZPH) width for that state is consistent with optical dynamics due to coupling with glasslike two-level systems of the protein. The ZPH width at 1.9 K is 0.037 cm-1. Satellite hole structure produced by burning in the above three states as well as their low linear pressures shift rates (about − 0.08 cm-1/MPa) indicate that the Chl a molecule of the subunit associated with them is weakly coupled to other Chl molecules. The linear pressure shift rates for the main Q y -absorption bands are also low. The shift rates appear to be dictated by protein−Chl interactions rather than excitonic couplings. Holes burned into the 650 nm absorption band reveal energy transfer times of 1 ps and ∼100 fs which are discussed in terms of time domain measurements of the Chl b → Chl a transfer rates (Connelly et al. J. Phys. Chem. B 1997, 101, 1902). The holewidths associated with burning into the 676 nm absorption band lead to Chl a → Chl a transfer times in the 6−10 ps range, in good agreement with the time domain values (Savikhin et al. Biophys. J. 1994, 66, 1597).