Understanding Two Different Structures in the Dark Stable State of the Oxygen-Evolving Complex of Photosystem†II: Applicability of the Jahn-Teller Deformation Formula.

Tanaka et al. (J. Am. Chem. Soc., 2017, 139, 1718) recently reported the three-dimensional (3D) structure of the oxygen evolving complex (OEC) of photosystem II (PSII) by X-ray diffraction (XRD) using extremely low X-ray doses of 0.03 and 0.12 MGy. They observed two different 3D structures of the CaMn4O5 cluster with different hydrogen-bonding interactions in the S1 state of OEC keeping the surrounding polypeptide frameworks of PSII the same. Our Jahn-Teller (JT) deformation formula based on large-scale quantum mechanics/molecular mechanics (QM/MM) was applied for these low-dose XRD structures, elucidating important roles of JT effects of the MnIII ion for subtle geometric distortions of the CaMn4O5 cluster in OEC of PSII. The JT deformation formula revealed the similarity between the low-dose XRD and damage-free serial femtosecond X-ray diffraction (SFX) structures of the CaMn4O5 cluster in the dark stable state. The extremely low-dose XRD structures were not damaged by X-ray irradiation. Implications of the present results are discussed in relation to recent SFX results and a blue print for the design of artificial photocatalysts for water oxidation.

Two Different Structures of the Oxygen-Evolving Complex in the Same Polypeptide Frameworks of Photosystem II.

The oxygen-evolving complex (OEC) forms the heart of photosystem II (PSII) in photosynthesis. The crystal structure of PSII from Thermosynechococcus vulcanus has been reported at a resolution of 1.9 Å and at an averaged X-ray dose of 0.43 MGy. The OEC structure is suggested to be partially reduced to Mn(II) by EXAFS and DFT computational studies. Recently, the "radiation-damage-free" structures have been published at 1.95 Å resolution using XFEL, but reports continued to appear that the OEC is reduced to the S0-state of the Kok cycle. To elucidate much more precise structure of the OEC, in this study two structures were determined at extremely low X-ray doses of 0.03 and 0.12 MGy using conventional synchrotron radiation source. The results indicated that the X-ray reduction effects on the OEC were very small in the low dose region below 0.12 MGy, that is, a threshold existed for the OEC structural changes caused by X-ray exposure. The OEC structures of the two identical monomers in the crystal were clearly different under the threshold of the radiation dose, although the surrounding polypeptide frameworks of PSII were the same. The assumption that the OECs in the crystal were in the dark-stable S1-state of the Kok cycle should be re-evaluated.

Three different mechanisms of energy dissipation of a desiccation-tolerant moss serve one common purpose: to protect reaction centres against photo-oxidation.

Three different types of non-photochemical de-excitation of absorbed light energy protect photosystem II of the sun- and desiccation-tolerant moss Rhytidium rugosum against photo-oxidation. The first mechanism, which is light-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is controlled by the protonation of a thylakoid protein. Other mechanisms are activated by desiccation. One of them permits exciton migration towards a far-red band in the antenna pigments where fast thermal deactivation takes place. This mechanism appears to be similar to a mechanism detected before in desiccated lichens. A third mechanism is based on the reversible photo-accumulation of a radical that acts as a quencher of excitation energy in reaction centres of photosystem II. On the basis of absorption changes around 800 nm, the quencher is suggested to be an oxidized chlorophyll. The data show that desiccated moss is better protected against photo-oxidative damage than hydrated moss. Slow drying of moss thalli in the light increases photo-protection more than slow drying in darkness.

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) in photosystem II.

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10 days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, flourescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.

Photoconversion mechanism of a green/red photosensory cyanobacteriochrome AnPixJ: time-resolved optical spectroscopy and FTIR analysis of the AnPixJ-GAF2 domain.

The photoconversion mechanism of a green/red sensory cyanobacteriochrome AnPixJ was studied. The phycocyanobilin-binding second GAF domain of AnPixJ of Anabaena sp. PCC 7120 was expressed in Escherichia coli cells. The His-tagged AnPixJ-GAF2 domain exhibited photoconversion between the green- and red-absorbing forms, APg(543) and APr(648), respectively. We detected four intermediate states in the photocycle between them, as follows: APr(648) → red light → APr(648)* → (with a rise time constant τ(r) of <100 ns) R1(650-80) (with a decay time constant τ(d) of <1 µs) → R2(610) (τ(d) = 920 µs) → APg(543) → green light → APg(543)* → (τ(r) < 50 ns) G1(570) (τ(d) = 190 µs) → G2(630) (τ(d) = 1.01 ms) → APr(648). These intermediates were named for their absorption peak wavelengths, which were estimated on the basis of the time-resolved difference spectra and global analysis of the time courses. The absorption spectrum of APr(648) resembles that of the Pr form of the phytochrome, while all the other states showed peaks at 530-650 nm and had wider bandwidths with smaller peak amplitudes. The fastest decay phases of fluorescence from APr(648)* and APg(543)* gave lifetimes of 200 and 42 ps, respectively, suggesting fast primary reactions. The APg(543)-minus-APr(648) difference FTIR spectrum in an H(2)O medium was significantly different from those reported for the Pfr/Pr difference spectra in phytochromes. Most of the peaks in the difference spectrum were shifted in the D(2)O medium, suggesting the high accessibility to the aqueous phase. The interactions of the phycocyanobilin chromophore with the surrounding amino acid residues, which are fairly different from those in the GAF domain of phytochromes, realize the unique green/red photocycle of AnPixJ.

Acceleration of electron-transfer-induced fluorescence quenching upon conversion to the signaling state in the blue-light receptor, TePixD, from Thermosynechococcus elongatus.

TePixD is a blue light using flavin (BLUF) protein of a thermophilic cyanobacterium, Thermosynechococcus elongatus. The fluorescence dynamics of TePixD was observed for the first time in both its dark-adapted and signaling (red-shifted) forms with a 200-fs time resolution. The fluorescence up-conversion setup was used in the time region up to 60 ps, and the streak-camera setup was used in the time region up to 1 ns. To avoid the accumulation of the red-shifted form by the exciting laser irradiation, the sample solution was circulated using a diaphragm pump. A handmade flow cuvette with a small cross section was used to achieve a fast flow of the solution in the excited region. The fluorescence decay times were unequivocally determined to be 13.6 and 114 ps for the dark-adapted form and 1.37 ps for the red-shifted form. The double-exponential fluorescence decay in the dark-adapted form suggested the coexistence of two conformations that have the 13.6- and 114-ps decay components, respectively. The single-exponential fluorescence decay in the red-shifted form suggested the elimination of heterogeneity in the conformation upon the light-induced conversion. The fast fluorescence-quenching components were almost eliminated in the mutant in which the conserved tyrosine Tyr8 is replaced by phenylalanine. Thus, the fluorescence quench was concluded to arise from the electron transfer from Tyr8, to the excited flavin chromophore. The 10-fold-faster quenching in the red-shifted form suggested the acceleration of the electron transfer. The faster decay time of 13.6 ps for the dark-adapted form was found to be almost temperature independent in the region from 10 to 40 degrees C. This suggested that the energy gap, DeltaG, in Marcus's electron-transfer theory is optimized to give the fastest rate. The acceleration of the electron transfer in the red-shifted form is interpreted to be due to the enhancement of the electronic-coupling factor between the donor and acceptor. A shortening of the Tyr8-flavin distance by 1.0-1.5 A was suggested if we adopt the empirical formula for the donor-acceptor distance dependence of the electron transfer rate.

Formation of interacting spins on flavosemiquinone and tyrosine radical in photoreaction of a blue light sensor BLUF protein TePixD.

Light-induced radicals were detected by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) in the BLUF-domain protein TePixD of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The illumination of TePixD at 5-200 K derived an EPR signal with a separation of 85 G between the main peaks around g = 2, showing a typical Pake's pattern of magnetic dipole-dipole interaction between two nearby radicals. Longer illumination induced an EPR signal at g = 2.0045, which was assigned as a neutral flavosemiquinone FADH(*). The FADH(*) formation occurred in parallel with a decrease in Pake's doublet. The Pake's doublet was not detected in a mutant TePixD protein in which a tyrosine residue was replaced with phenylalanine (Y8F protein). A pulsed ENDOR study suggested that the Pake's doublet had arisen from the interaction between a neutral flavosemiquinone radical and a neutral tyrosine radical, i.e., the FADH(*)-Y8(*) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 A shorter than that calculated from the X-ray crystallography structure in the dark-adapted state, suggesting the modification of the protein conformation in the photoinduced FADH(*)-Y8(*) state. The Pake's doublet signal was detected by 10 K illumination in the sample which was immediately frozen after 273 K illumination, corresponding to the red-shifted state F(490). On the other hand, the signal was not detected in the sample which was incubated for 10 min at 273 K in the dark after 273 K illumination, corresponding to the dark-adapted state D(471). In the sample annealed at 160 K for 10 min after 160 K illumination, corresponding to the partially red-shifted state J(11), the Pake's doublet signal was detected by the 10 K illumination. On the basis of these observations, we concluded that the interaction with the FADH(*)-Y8(*) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.

A novel photoactive GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red photoconversion.

We report the discovery of a novel cyanobacteriochrome, the green/red photoreceptor AnPixJ (All1069), isolated from the heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120. Cyanobacteriochromes are a recently emerging tetrapyrrole-based photoreceptor superfamily that are distantly related to the conventional red/far-red photoreceptor phytochromes (Phys). The chromophore-binding domains of AnPixJ produced in cyanobacterial and Escherichia coli cells both showed a reversible and full photoconversion between a green-absorbing form (lambda(max)=543 nm) and a red-absorbing form (lambda(max)=648 nm). Denaturation analysis revealed that the green-absorbing form and the red-absorbing form covalently ligated phycocyanobilin with E-configuration and Z-configuration at the C15C16 double bond, respectively. Time-resolved spectral analysis showed the formation of the first intermediate state peaking at 680 nm from the dark-stable red-absorbing form. This step resembles the first photoconversion step from the red-absorbing form to the red-shifted lumi-R intermediate state of the Phys. These results suggest that the Pr of AnPixJ is almost equivalent to that of the Phys and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the Phys, but is finally photoconverted to the unique green-absorbing form.

Photoreactions of Tyr8- and Gln50-mutated BLUF domains of the PixD protein of Thermosynechococcus elongatus BP-1: photoconversion at low temperature without Tyr8.

We studied the photoreaction of a blue-light sensor PixD protein of Thermosynechococcus elongatus that has the blue-light-using flavin (BLUF) domain. The Tyr8 and Gln50 residues of the protein were modified to phenylalanine, alanine, or asparagine (Y8F, Y8A, Q50N, and Q50A) by site-directed mutagenesis. The following results were obtained. (1) At room temperature, blue-light illumination induced the red shift of the absorption bands of flavin in the wild-type (WT) protein but not in the Y8F, Y8A, Q50A, and Q50N mutant proteins, as reported [Okajima, K., et al. (2006) J. Mol. Biol. 363, 10-18]. (2) At 80 K, neither the Q50N nor the Q50A mutant protein accumulated the red-shifted form. (3) At 80 K, the Y8F protein photoaccumulated the red-shifted forms to an extent that was half that in the WT protein at a 43-fold slower rate, and the Y8A protein to the one-fourth the extent at a 137-fold slower rate. (4) The red-shifted form in the Y8F protein was stable below 240 K and became unstable above 240 K in the dark. (5) The illumination of the Y8F protein at 150 K accumulated the red-shifted form at the beginning, and the prolonged illumination accumulated the flavin anions by the secondary photoreaction. (6) The results indicate that Tyr8 is not indispensable for the accumulation of the red-shifted form at least at 80 K. (7) Photoconversion mechanisms in the WT and Tyr8-mutated proteins are discussed in relation to the schemes with and without the electron transfer between Tyr8 and flavin in the first step of the photoconversion.

Two intermediate states I and J trapped at low temperature in the photocycles of two BLUF domain proteins of cyanobacteria Synechocystis sp. PCC6803 and Thermosynechococcus elongatus BP-1.

We identified the two intermediate states, I and J, that are common in the photocycles of the cyanobacterial BLUF (sensor of Blue Light Using Flavin) domain proteins of Slr1694 of Synechocystis sp. PCC6803 and Tll0078 of Thermosynechococcus elongatus BP-1 by analyzing the absorption spectra at 5 K. Illumination at 5 K accumulated intermediate forms (designated as I5 and I9), which showed 5 and 9 nm redshifts of the absorption bands of flavin in the Tll0078 and Slr1694 proteins, respectively. I5 (I9) was converted into the next intermediate, which have 11 nm (14 nm) red-shifted absorption bands J11 (J14) after dark annealing at 230 K (240 K). Further dark annealing at 280 K (270 K) of J11 (J14) produced the signal-transmitting final form F490 (F495), with a small increase in the absorption at around 490 nm (495 nm). The results indicate that the BLUF proteins of Tll0078 and Slr1694 exhibit the common photocycle of D471 (D467) --> I5 (I9) --> J11 (J14) --> F490 (F495) at low temperature. The transition temperatures for these intermediate forms differ for two proteins. The amount of I5 (I9) accumulated at 5 K was small and increased at a higher temperature, suggesting heterogeneity of the protein structure that determines the reaction pathway.

Fate determination of the flavin photoreceptions in the cyanobacterial blue light receptor TePixD (Tll0078).

PixD (Tll0078, Slr1694) is a BLUF (sensor of blue light using FAD)-type blue light receptor protein of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and the mesophilic cyanobacterium Synechocystis sp. PCC 6803. BLUF protein is known to show light-induced approximately 10 nm red shift of flavin absorption that is coupled with strengthening of the hydrogen bond between the O(4) of the isoalloxazine ring and a certain amino acid residue. According to the 3D structure of TePixD we determined, O(4) of the ring is linked to Gln50 and Asn32. A survey of flavin-interacting residues by site-directed mutagenesis showed that Gln50 but not Asn32 is essential for the normal red-shifting photoreaction. Here, we further studied the role of Gln50 and its close neighbor Tyr8. All the mutated proteins of Gln50 and Tyr8 (Q50A, Q50N, Y8A and Y8F) lost the normal red-shifting photoreaction. Y8A, Y8F and Q50N, instead, showed a light-induced flavin triplet state and a low yield of subsequent flavin reduction that is analogous to the photocycle of the LOV (light-oxygen-voltage-sensing) domain of phototropins, while Q50A did not. Fourier-transform infrared (FT-IR) analysis of N32A showed that O(4) of the ring is hydrogen-bonded to Asn32 both in the light and dark. These results, together with the 3D structure, indicate that the hydrogen bond network of Tyr8-Gln50-O(4)/N(5) (flavin) is critical for the light reaction of the BLUF domain. Based on the structural and functional similarities of the BLUF and the LOV domain of phototropins, we propose that the interaction between apoprotein and N(5) of flavin determines the photoreaction of the flavin-binding sensors.

Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria.

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.

Primary intermediate in the photocycle of a blue-light sensory BLUF FAD-protein, Tll0078, of Thermosynechococcus elongatus BP-1.

Proteins with a BLUF (sensor of blue light using flavin adenine dinucleotide) domain represent a newly recognized class of photoreceptors that is widely distributed in the genomes of photosynthetic bacteria, cyanobacteria, and Euglena. Recently, Okajima et al. [Okajima, K., Yoshihara, S., Geng, X., Katayama, M. and Ikeuchi, M. (2003) Plant Cell Physiol. 44 (Suppl), 162] purified BLUF protein Tll0078 encoded in the genome of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by expressing the protein in Escherichia coli. We investigated the photocycle of Tll0078 by measuring the picosecond fluorescence kinetics, transient absorption changes, and the UV-visible absorption spectra at 10 to 330 K. The absorption spectrum of the FAD moiety of Tll0078 showed a 10-nm red shift upon illumination at 278-330 K. The quantum efficiency of the formation of the red-shifted form was 29%. Illumination at 10 K, on the other hand, caused only a 5-nm red shift in about one-half of the protein population. The 5-nm-shifted form was stable at 10 K. The 5-nm red-shifted form was converted into the 10-nm red-shifted form at 50-240 K upon warming in the dark. At room temperature, the 10-nm red-shifted final product appeared within 10 ns after laser flash excitation. The lifetime of the fluorescence of FAD was found to be 120 ps at room temperature. These results reveal a fast and efficient photoconversion process from the singlet-excited state to the final product at room temperature. A photocycle of BLUF protein is proposed that includes the 5-nm red-shifted intermediate form as the precursor for the 10-nm red-shifted final product. The temperature dependence of each step of the photocycle is also discussed.