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A detailed characterization of electron-phonon and electron-vibrational coupling strengths of a weakly coupled chlorophyll transition of the antenna complex CP29 of photosystem II of green plants at 4.5 K is described.

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the [Mn.sub.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.sub.4]([III.sub.2], [IV.sub.2]) oxo-bridged [Mn.sub.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. manganese | oxygen evolution | water oxidation | x-ray spectroscopy

The overexpression of Escherichia coli and the characterization of the gene product by biochemical and spectroscopic methods are described. recPsbO was titrated in the presence of Ca(super 2+), followed by UV-circular dichroism spectropolarimetry (CD) and Fourier transform infrared spectroscopy in order to elucidate probable Ca(super 2+) binding site(s), but no significant changes in the secondary structure could be observed.

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) are isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-D-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical centrifugation (AUC). The results are discussed from a crystallization point of view.

Vibrational dynamics of the light-harvesting complex II (LHC II) from spinach was investigated by quasi- and inelastic neutron scattering (QENS and INS) at three different temperatures of 80, 160, and 285 K. QENS/INS spectra of solubilised LHC II and of the corresponding buffer solution were obtained separately and exhibit characteristic inelastic features. After subtraction of the buffer contribution, the INS spectrum of LHC II reveals a distinct Boson peak at ∼ 2.5 meV at 80 K that shifts towards lower energies if the temperature is increased to 285 K. This effect is interpreted in terms of a “softening” of the protein matrix along with the dynamical transition at ∼ 240 K. Our findings indicate that INS is a valuable method to obtain the density of vibrational states not only at cryogenic, but also at physiological temperatures.

Results reveal pronounced excitonic interactions between chlorophyll a and b molecules, suggesting that the spectral heterogeneity of a minor photosystem II antenna complex includes contribution of the chlorophyll molecules as well as the contribution from the pigment-protein interactions.

A study was conducted to examine the association between the binding of dicyclohexylcarbodiimide (DCCD) and the inhibition of chlorophyll fluorescence quenching in various light-harvesting system of photosystem II (PSII) preparations by DCCD. Results indicated that a number of amino acid residues support a critical role in regulating PSII function. Findings also showed that DCCD specifically and covalently bounded to light harvesting complexes.

The kinetic H/D isotope exchange effects on the univalent oxidation steps of the water oxidizing complex (WOC) in photosystem II membrane fragments and PSII core complexes from spinach as well as the temperature dependence of the reactions were analyzed using flash-induced absorption changes measured at different temperatures. Results have shown that the activation energies of each redox step in the WOC were dependent on the redox state and that deuteron substitutions cause only minor changes.

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. [ABSTRACT FROM AUTHOR]

Persistent nonphotochemical hole burning and delta-FLN spectra obtained at 4.5 K are reported for monomeric chlorophyll (Chl) a/b light-harvesting complexes of photosystem II (LHC II) of green plants. The hole burned spectra of monomeric LHC II appear to be similar to those obtained before for trimeric LHC II (Pieper et al. J. Phys. Chem. B 1999, 103, 2412). They are composed of three main features: (i) a homogeneously broadened zero-phonon hole coincident with the burn wavelength, (ii) an intense, broad hole in the vicinity of approximately 680 nm as a result of efficient excitation energy transfer to a low-energy trap state, and (iii) a satellite hole at approximately 649 nm which is correlated with the low-energy 680 nm hole. Zero-phonon hole action spectroscopy reveals that the low-energy absorption band is located at 679.6 nm and possesses a width of approximately 110 cm(-1) which is predominantly due to inhomogeneous broadening at 4.5 K. The electron-phonon coupling of the above-mentioned low-energy state(s) is weak with a Huang-Rhys factor S in the order of 0.6 and a peak phonon frequency (omega(m)) of approximately 22 cm(-1) within a broad and strongly asymmetric one-phonon profile. The resulting Stokes shift 2S omega(m) of approximately 26.4 cm(-1) readily explains the position of the fluorescence origin band at 680.8 nm. Thus, we conclude that the 679.6 nm state(s) is (are) the fluorescent state(s) of monomeric LHC II at 4.5 K. The absorption intensity of the lowest Q(y) state is shown to roughly correspond to that of one out of the eight Chl a molecules bound in the monomeric subunit. In addition, the satellite hole structure produced by hole burning within the 679.6 nm state is weak with only one shallow satellite hole observed in the Chl b spectral range at 648.8 nm. These results suggest that the 679.6 nm state is widely localized on a Chl a molecule, which may belong to a Chl a/b heterodimer. These characteristics are different from those expected for Chl a612, which has been associated with the fluorescent state at room temperature. Alternatively, the 679.6 nm state may be assigned to Chl a604, which is located in a cluster with several Chl b molecules resulting in a relatively weak excitonic coupling.