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DNA photolyase is a pyrimidine-dimer repair enzyme that uses visible light. Photolyase generally contains two chromophore cofactors. One is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. The other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. Photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. The native structures of both types of photolyases have already been determined, but the mechanism of substrate recognition remains largely unclear because of the lack of structural information regarding the photolyase-substrate complex. Photolyase from Thermus thermophilus, the first thermostable class I photolyase found, is favorable for function analysis, but even the type of the second cofactor has not been identified. Here, we report the crystal structures of T. thermophilus photolyase in both forms of the native enzyme and the complex along with a part of its substrate, thymine. A structural comparison with other photolyases suggests that T. thermophilus photolyase has structural features allowing for thermostability and that its light-harvesting cofactor binding site bears a close resemblance to a deazaflavin-type photolyase. One thymine base is found at the hole, a putative substrate-binding site near the catalytic cofactor in the complex form. This structural data for the photolyase-thymine complex allow us to propose a detailed model for the pyrimidine-dimer recognition mechanism.

EPR measurements of different states from S(sub)0 to S(sub)3 in histidine-tagged Synechococcus elongatus suggest that histidine-tag per se has no influence on the EPR of the manganese-cluster of photosystem II. Results demonstrate that perpendicular and polarization EPR yield signals for the S(sub)3 state, a first time record for the cyanobacteria.

A study was conducted to identify the histidine signals in the S2/S1 Fourier transformation infrared difference spectrum using photosystem (PS) II membranes from globally (super 15)N-labeled spinach and PS II core complexes from Synechocytis cells in which both the imidazole nitrogens of histidine were selectively labeled with (super 15)N. Moreover, model compounds of histidine side chains were measured to provide infrared markers of protonation forms and hydrogen bonding interaction of the imidazole ring. The interaction and residue of the histidine residue in the oxygen-evolving complex was described with reference to the markers.

NDFS and I6, two phototolerant mutants, were examined by thermoluminescence measurements to track the thermodynamics of redox equivalents contained on the donor receptor and acceptor sides of photosystem II. The expression mechanisms of phototolerance and the nonphotobleaching phenotype of these mutants are also analyzed. Implications of the results, such as decreased equilibrium constants for sharing an electron between the primary, QA, and secondary acceptor plastoquinones, QB, are also discussed.

Interactions of the primary quinone receptor Q(sub A) of photosystem II (PS II) with neighboring amino acid residues were investigated by examining the Fourier transform infrared difference spectra of Q(sub A) upon its photoreduction. Findings showed that a His side chain is strongly hydrogen bonded to Q(sub A) through the imidazole NH, indicating that the Q(sub A)-His interaction in the bacterial reaction center is conserved in PS II.

Simulation studies of multiline electron paramagnetic resonance spectra using relatively large anisotropic ultra-fine tensors and an anisotropic g-tensor for the Mn cluster in partially oriented photosystem (PS) II membranes were derived. This Mn cluster is surmised to provide a catalytic site for water oxidation. Results provide data on the orientation of the principle axes of the g-tensor of the Mn cluster with respect to the normal of thylakoid membrane and the relative arrangement of the principle axes of the ultra-fine tensor of a Mn(III) ion and the g-tensor of the cluster.

A functional characterization of Synechocystis sp. PCC 6803 deltapsbU and deltapsbV mutants was implemented through measurements of thermoluminescence, flash oxygen yield and dark-reactivation. The destabilization of PSII complex in vivo resulted in substantial loss of oxygen evolution upon deletion of cyt c-550. The results suggested that cytochrome c-550 has an important role in cyanobacterial oxygen evolution.

The presence of a structural coupling between redox-active tyrosine 161 of the D1 polypeptide (Y(sub z)) and the manganese (Mn) cluster in an intact oxygen-evolving complex was investigated through Fourier transform infrared spectroscopy. The linkage coupling formed a proton transfer pathway and an indirect proton transfer from substrate water to Yz. In S-state transition, the hydrogen bond between Yz and Mn catalyzed the electron transfer from the Mn cluster to Yz.

Light-induced Fourier transform spectroscopy was utilized to investigate the structure and molecular interactions of cofactors in photosynthetic reaction centers (RC). The observed frequencies indicated that S-F formed a strong hydrogen bond which was further strengthened following P798+ formation. Measurement of the polarized FTIR difference spectra with an oriented membrane film aided the assignments of the C=O bands.

The mechanisms that mediate the photoactivation and dark-inactivation of Rhodococcus sp. N-771 nitrile hydratase (NHase) were analyzed to investigate the role of the iron(III) center in the binding of endogenous NO. Analysis of the inactive NHase that was irradiated in the presence of Fe(II) ions and N-methyl-D-glucamine dithiocarbamate (MGD) indicated the role of photoactivation in releasing NO from the enzyme. The enzymatic activity of the enzyme was modulated by NO binding and photodissociation.

Resonance Raman spectra of the nitrile hydratase (NHase) from Rhodococcus sp. N-771 were measured with 413 nm excitation at 85 K to directly determine the bonding between nitric oxide (NO) and the iron atom and the reaction of NO upon photoactivation. Two prominent bands at 592 and 570/cm. were seen in the inactive form and both were completely lost upon photoactivation. Findings showed that NO is bound to the non-heme iron in the inactive NHase and its photodissociation activates the enzyme.

Light-induced Fourier transform infrared difference spectra of P840 upon its oxidation were measured with the reaction center complex from the green sulfur bacterium Chlorobium tepidium. A broad band centered near 2500/cm was seen in P(sub 840)(super +), which is comparable to the band near 2600/cm previously seen in P(sub 870)(super +) of purple bacteria and assigned to the electronic transition in the bacteriochlorophyll a dimer. The intensity of this electronic band found in P(sub 840)(super +) was about the same as that in P(sub 870)(super +).

Cytochrome (cyt) c-550 is an extrinsic protein, analogous to the 33 kDa protein, which independently supports oxygen evolution in cynobacterial photosystem II and maintains photoautotrophic development. A double-deletion mutant devoid of both c-550 and the 33 kDa protein by simultaneous inactivation of the psbO and psbV genes in Synechocystis sp. PCC 6803 reveals inhibited oxygen-producing ability.

Thermoluminescence and flash-induced oxygen evolution of the water-oxidizing complex (WOC) were measured to investigate the deactivation/photoactivation processes and determine the role of the chlorophyll alpha-binding photosystem II protein CP47 in creating a functional WOC. The WOC became inactive during long periods of dark incubation in a number of mutants, while the WOC of the wild-type strain continued to exhibit activity. Results affirmed the important role of the loop E of CP47 protein in the stability and photoactivation of the WOC.

Site-directed mutations at three amino acid positions in the manganese-stabilizing protein (MSP) to determine the function of putative intermolecular binding residues in the binding between the N-terminal region of MSP and the photosystem II reaction center of cyanobacteria indicate that replacement of the aspartate with lysine does not affect MSP accumulation. Genetic distortions in the intramolecular disulfide linkage inhibit MSP accumulation. Substitution of aspartate 159 with asparagine results in an enhancement of the thermoluminescence properties and H2O-oxidation activity of the cyanobacteria.

A study of autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions or a single-site mutation within the large, lumen-exposed loop of the chlorophyll a-binding photosystem II core protein, CP47, through measurement of thermoluminescence, oxygen yield and fluorescence quantum yield, reveals that deletion of charged and conserved amino acids between residues 370 and 390 decreases the binding affinity of the extrinsic PS II-O protein to photosystem II. Marked differences with PS II-O deletion mutants, with respect to Ca2+ requirement and flash-induced pattern of oxygen evolution, exist.

A study using release-reconstitution and thermoluminescencemethods was conducted on the binding and functional characteristics of cytochrome (cyt) c-550 and a 12-kDa protein in cyanobacterial photosystem (PS) II. Results reveal that, in the absence of the 12- and 33-kDa proteins, a rebinding occurred between cyt c-550 and PSII. In addition, it was found that the 12-kDa protein rebound to PSII only in the presence of the 33-kDa protein and cyt c-550 and that the O2-evolving activity of PSII washed with CaCl2 couldnot be restored by reconstitution with cyt c-550 or the 12-kDa protein unless the 33-kDa protein was present.

A study was conducted on the modulation of the manganese cluster found in photosystem II membranes depleted of Ca2+. Calcium depletion was found to produce a modified S2 state. The characteristics of the Ca2+-depleted S2 state was further attributed to the association of the 24-kilodalton extrinsic protein to photosystem II. The results also suggest that the extrinsic protein controls the structure and function of the manganese cluster in the absence of functional calcium.

A study done on the photosystem II of the Synechocystis sp. PCC 6803 mutant involved calculating the concentration of photochemically active PSII reactions in wild and mutant types as well as characterizing O2 evolution in the mutants. It was shown that S2 and S3 mutants were more stable to recombination with negative charges compared to the wild types. In addition, it was further confirmed that the loss of manganese-stabilizing proteins leads to retardation of one or more of the S-state transitions and a reduced efficiency in the utilization of oxidizing equivalents generated at the reaction center.

The molecular structure and structural changes of manganese (Mn) cluster in oxygen-evolving photosystem II (PSII) were examined by the use of light-induced Fourier transform infrared (FT-IR) difference spectroscopy. The use of an exogenous electron acceptor to trypsin-treated PSII membranes and light illumination at 250K freed the S2/S1 difference spectrum from contributions by the acceptor-side signals. This S2/S1 spectrum demonstrated the structural transformation within the Mn cluster upon the S1 to S2 transition. The success of this method indicated its potential use for further molecular analyses of the Mn structure and the O2-evolving mechanism.

Eight differently substituted 1,4-benzoquinones were prepared and their effects on the quantum yield of photoactivation of oxygen evolution in wheat photosystem II (PSII) membranes depleted of the Mn cluster were determined. The results suggest that the enhancement of activity recovery is caused by rapid oxidation by the benzoquinones of the reduced form of the quinone acceptors in PSII. Notably, direct evidence that two is the minimum quantum requirement for photoactivation is presented.

Atmospheric oxygen is a result of water cleavage by oxygenic photosynthesis in plants. The fundamental view of the mechanism of photosynthetic water oxidation was introduced by Joliot et al., who [...], X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn K-edge showed a period-four oscillation dependent on cycling of the Joliot-Kok's oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the [S.sub.0]-to-[S.sub.1], [S.sub.1]-to-[S.sub.2], and [S.sub.2]-to-[S.sub.3] transitions and then reduced upon the [S.sub.3]-to-[S.sub.0] transition that releases molecular oxygen.

The X-ray absorption near-edge structure (XANES) spectroscopy data indicate that the K-edge energy does not propose any changes in the ligation structure of the Mn cluster. The depletion of Cl(super -) does not cause any considerable structural change of the Mn cluster in the photosynthetic water oxidation catalyzed by the tetranuclear Mn cluster lying in the photosystem II.

Research was conducted to examine the structure and interaction of cofactors in photosynthetic proteins using light-induced Fourier transform infrared difference spectroscopy. The measurement of the spectrum of P680 was performed upon its photooxidation in the requency region of 1000-3000/cm. Results demonstrate that a positive charge on P680+ is delocalized over the accessory chlorophyll (Chl) molecules. Findings also suggest that the triplet state generated at low temperatures resides on a Chl molecule different from the Chls that possess a positive charge in P680+.