The sensor of blue-light using FAD (BLUF) domain is the flavin-binding fold categorized to a new class of blue-light sensing domain found in AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis, but little is known concerning the mechanism of blue-light perception. An open reading frame slr1694 in a cyanobacterium Synechocystis sp. PCC6803 encodes a protein possessing the BLUF domain. Here, a full-length Slr1694 protein retaining FAD was expressed and purified and found to be present as an oligomeric form (trimer or tetramer). Using the purified Slr1694, spectroscopic properties of Slr1694 were characterized. Slr1694 was found to show the same red-shift of flavin absorption and quenching of flavin fluorescence by illumination as those of AppA. These changes reversed in the dark although the rate of dark state regeneration was much faster in Slr1694 than AppA, indicating that Slr1694 is a blue-light receptor based on BLUF with the similar photocycle to that of AppA. The dark decay in D(2)O was nearly four times slower than in H(2)O. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was applied to examine the light-induced structure change of a chromophore and apo-protein with deuteration and universal (13)C and (15)N isotope labeling. The FTIR results indicate that light excitation induced distinct changes in the amide I modes of peptide backbone but relatively limited changes in flavin chromophore. Light excitation predominantly weakened the C(4)=O and C(2)=O bonding and strengthened the N1C10a and/or C4aN5 bonding, indicating formational changes of the isoalloxazine ring II and III of FAD but little formational change in the isoalloxazine ring I. The photocycle of the BLUF is unique in the sense that light excitation leads to the structural rearrangements of the protein moieties coupled with a minimum formational change of the chromophore.

Ultraviolet (UV) light induces specific mutations in the cellular and skin genome such as UV-signature and triplet mutations, the mechanism of which has been thought to involve translesion DNA synthesis (TLS) over UV-induced DNA base damage. Two models have been proposed: "error-free" bypass of deaminated cytosine-containing cyclobutane pyrimidine dimers (CPDs) by DNA polymerase η, and error-prone bypass of CPDs and other UV-induced photolesions by combinations of TLS and replicative DNA polymerases--the latter model has also been known as the two-step model, in which the cooperation of two (or more) DNA polymerases as misinserters and (mis)extenders is assumed. Daylight UV induces a characteristic UV-specific mutation, a UV-signature mutation occurring preferentially at methyl-CpG sites, which is also observed frequently after exposure to either UVB or UVA, but not to UVC. The wavelengths relevant to the mutation are so consistent with the composition of daylight UV that the mutation is called solar-UV signature, highlighting the importance of this type of mutation for creatures with the cytosine-methylated genome that are exposed to the sun in the natural environment. UVA has also been suggested to induce oxidative types of mutation, which would be caused by oxidative DNA damage produced through the oxidative stress after the irradiation. Indeed, UVA produces oxidative DNA damage not only in cells but also in skin, which, however, does not seem sufficient to induce mutations in the normal skin genome. In contrast, it has been demonstrated that UVA exclusively induces the solar-UV signature mutations in vivo through CPD formation.

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The imidazole group of a histidine side chain has four different protonation forms, i.e., the two neutral tautomers (N1-and N3-protonated forms), imidazolium cation, and imidazolate anion. Owing to the presence of these convertible protonation forms, histidine plays important roles in proton-transfer reactions in various enzymes. Vibrational spectroscopy is one of the most powerful methods to study the protonation state of histidine in proteins. For systematic investigation of IR and Raman markers of the protonation state of histidine, we have performed ab initio normal-mode calculations using the density function theory (DFT) method for all of the four protonation forms of 4-methylimidazole (a simple model compound of a histidine side chain) and their N-deuterated analogues. FTIR and Raman spectra of all of these compounds were measured, and the observed bands were assigned according to the calculated frequencies and intensities. Differences in the optimized geometries and changes in the vibrational couplings explained the differences in band frequencies and N-deuteration shifts among the protonation forms. These analyses provided theoretical bases for the IR and Raman markers of the protonation state, including known markers, such as the C4C5 stretching and the C5N1 stretching bands, as well as some new potential markers.

AppA is a new class blue-light receptor controlling photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides and retains a characteristic flavin adenine dinucleotide (FAD)-binding domain named the "sensor of blue light using FAD" (BLUF). AppA functions as an antirepressor controlling transcription of photosynthesis genes through the direct association with a transcriptional repressor PpsR in a blue-light-dependent manner [Masuda and Bauer (2002) Cell 110, 613-623]. Illumination of AppA induces a red shift in the UV-visible absorption of FAD, which results in a signaling state of AppA. Light-induced Fourier transform infrared (FTIR) difference spectrum of the AppA BLUF domain showed relatively simple features, which were mainly composed of two sets of derivative-shaped sharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm(-)(1). We have developed an in vitro reconstitution method, by which a fully functional BLUF domain was reconstituted from free FAD and an apoprotein for the BLUF domain of AppA. An AppA BLUF domain that consisted of an apoprotein isotopically labeled with (13)C and unlabeled FAD was constituted using this method, and hydrated and deuterated samples were applied to FTIR spectroscopic analyses. When the spectra for the reconstituted domain were compared with those for uniformly (15)N- and (13)C-labeled or deuterated domains as well as for the unlabeled domain, the IR bands responsible for the light-induced changes in the FAD chromophore and apoprotein were identified. Unexpectedly, the light-induced spectrum of the unlabeled BLUF domain of AppA was predominantly composed of multiple apoprotein bands, while a C(4)=O stretching of an isoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relatively large structural changes occur in the protein backbone of the BLUF domain of AppA upon illumination. These changes were discussed in relation to the mechanistic role of the BLUF domain in the process of blue-light perception by AppA.

Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. AppA functions as a transcriptional antirepressor, controlling the photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. Upon light absorption, AppA undergoes a photocycle with a signaling state, which exhibits an approximately 10 nm red shift in the UV-vis absorption spectrum. We have characterized light-dependent changes in the active site of an AppA BLUF domain by Raman spectroscopy. The present study has found that altered chromophore-protein interactions, including a hydrogen bond at the C4=O position and structural changes around the N10-ribityl side chain, are key events in this activation process. These structural alterations are proposed to be responsible for the transmission of the light signal in the BLUF domain. This is the first report on a signaling-state Raman spectrum of a blue-light photoreceptor with a flavin chromophore.

Treatment of spinach PS II membraneswith a citrate solution at pH 3.0 totally inactivated 0, evolution concomitant with a 50% decrease in Ca abundance.Notably, neither the abundance of Mn and extrinsic proteins nor the activity of DPC photooxidation was at all affected by the treatment. The treated membranes evolved 0, at a high rate in the presence *+ of exogenous Ca , but the activity was sensitive to EDTA. However, when the treated membranes were incubated with Ca* + for a few tens of minutes, the O,-evolving activity became EDTA-resistant, suggesting a firm re-ligation of Ca* + to the Ca-binding site. It was indicated that spinach PS II contains two Ca atoms per reaction center, and that the low pH citrate treatment selectively removes one of the two Ca atoms that is specifically functional for 0, evolution, even in the presence of all three extrinsic proteins.Oxygen evolution; Photosystem II membrane; Ca2+ extraction; Low pH treatment; 16 kDa protein; 24 kDa protein; 33 kDa protein