Takumi Noguchi

X-ray Detection of the Period-Four Cycling of the Manganese Cluster in Photosynthetic Water Oxidizing Enzyme.

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 Kedge showed a period-four oscillation dependent on cycling of the Joliot-Koks oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S0-to-S1, S1-to-S2, and S2-to-S3 transitions and then reduced upon the S3-to-S0 transition that releases molecular oxygen.

Direct detection of a carboxylate bridge between Mn and Ca2+ in the photosynthetic oxygen-evolving center by means of Fourier transform infrared spectroscopy

Abstract Calcium is an indispensable cofactor for photosynthetic oxygen evolution. We have studied structural relevance of Ca2+ to the oxygen-evolving center (OEC) of Photosystem II (PS II) by means of Fourier transform infrared (FTIR) spectroscopy. The single-pulse induced FTIR difference spectra of PS II membranes reflecting solely the structural changes of OEC between in the S1 and S2 states were measured by controlling the redox potential and pH of the buffer. Comparison between the two S2/S1 difference spectra using untreated and Ca2+-depleted PS II membranes showed that the negative bands at 1560 and 1403 cm−1 (belonging to S1) and the positive bands at 1587 and 1364 cm−1 (belonging to S2) were lost upon Ca2+ depletion. These bands were assigned to the asymmetric (higher frequency bands) and symmetric (lower frequency bands) COO− stretching modes of a certain carboxylate group in Asp, Glu or the C-termini, based on the infrared data of 20 amino acids and the S2/S1 spectra of 15N-labeled PS II membranes. The frequency differences of the asymmetric and symmetric COO− bands, i.e., 157 cm−1 for S1 and 223 cm−1 for S2, indicated that this carboxylate group possesses the structure of bridging bidentate coordination in the S1 state and that of unidentate coordination in the S2 state. Taking together the observation of disappearance of these bands upon Ca2+ depletion, it was concluded that (i) this carboxylate serves as a bridging ligand between the redox-active Mn and the Ca2+ ions, (ii) upon S2 formation, the coordination bond of this carboxylate to Ca2+ is selectively broken, and (iii) upon depletion of Ca2+, this carboxylate ligand is liberated even from the Mn ion. Along with the changes of COO− bands, several intense bands in 1680–1630 cm−1, which were assigned to the amide I modes of backbone amide groups, were lost upon Ca2+ depletion. This indicates that some perturbations on the protein conformations around the Mn-cluster induced by the S2 formation require the presence of Ca2+ in OEC. Possible roles of Ca2+ in the oxygen-evolving reactions are discussed based on these findings.

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, ‘distorted-chair’ form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’ structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium

The composition of photosystem II (PSII) in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017 was investigated to enhance the general understanding of the energetics of the PSII reaction center. We first purified photochemically active complexes consisting of a 47-kDa Chl protein (CP47), CP43′ (PcbC), D1, D2, cytochrome b559, PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 ± 7 Chl d, 3.0 ± 0.4 Chl a, 17 ± 3 α-carotene, and 1.4 ± 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FTIR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. The primary electron acceptor was shown by photoaccumulation to be Phe a, and its potential was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Taking into account the reported downward shift in the potential of the special pair of photosystem I (P740) in A. marina, our findings lend support to the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.

Monitoring proton release during photosynthetic water oxidation in photosystem II by means of isotope-edited infrared spectroscopy.

In photosynthetic water oxidation performed in the water oxidizing center (WOC) of photosystem II (PSII), two water molecules are converted into one oxygen molecule and four protons through a light-driven cycle of intermediates called S states (S(0)-S(4)). To understand the molecular mechanism of water oxidation and the chemical nature of substrate intermediates, it is essential to determine the stoichiometry of proton release from substrate water at individual S-state transitions. In this study, we have monitored proton release during water oxidation by means of isotope-edited Fourier transform infrared (FTIR) spectroscopy. FTIR difference spectra upon successive flash illumination were measured using PSII core complexes from a thermophilic cyanobacterium Thermosynechococcus elongatus, which were suspended in a high concentration (200 mM) Mes buffer at pH 6.0. The spectra involved, in addition to protein bands, the bands of the Mes buffer that trapped virtually all protons from the WOC. Mes-only signals were extracted by subtracting the spectra measured in deuterated-Mes (Mes-d(12)). The flash-number dependence of the intensity increase of the isotope-edited Mes signal showed a clear period-four oscillation. By simulating the oscillation with different assumptions about miss factors, the proton release pattern was estimated to be 0.8-1.0:0.2-0.3:0.9-1.2:1.5-1.6 for the S(0)-->S(1)-->S(2)-->S(3)-->S(0) transitions. The effect of H/D exchange on the COOH region of proteins in FTIR difference spectra of the S-state cycle showed that protonation/deprotonation of carboxylic groups contributed little to the observed proton release pattern. Together with the present and previous FTIR results suggesting no involvement of also His and Cys side groups, it was concluded that proton release from substrate water takes place with a 1:0:1:2 stoichiometry, which is perturbed by partial protonation/deprotonation of side groups probably of Arg, Lys, or Tyr located nearby the WOC.

Structure of a histidine ligand in the photosynthetic oxygen-evolving complex as studied by light-induced fourier transform infrared difference spectroscopy.

Fourier transform infrared (FTIR) signals of a histidine side chain were identified in flash-induced S(2)/S(1) difference spectra of the oxygen-evolving complex (OEC) of photosystem II (PS II) using PS II membranes from globally (15)N-labeled spinach and PS II core complexes from Synechocystis cells in which both the imidazole nitrogens of histidine were selectively labeled with (15)N. A negative band at 1113-1114 cm(-1) was downshifted by 7 cm(-1) upon both global (15)N-labeling and selective [(15)N]His labeling, and assigned to the C-N stretching mode of the imidazole ring. This band was unaffected by H-D exchange in the PS II preparations. In addition, several peaks observed at 2500-2850 cm(-1) all downshifted upon global and selective (15)N-labeling. These were ascribed to Fermi resonance peaks on a hydrogen-bonding N-H stretching band of the histidine side chain. FTIR measurements of model compounds of the histidine side chain showed that the C-N stretching band around 1100 cm(-)(1) can be a useful IR marker of the protonation form of the imidazole ring. The band appeared with frequencies in the following order: Npi-protonated (>1100 cm(-1)) > imidazolate > imidazolium > Ntau-protonated (<1095 cm(-1)). The frequency shift upon N-deuteration was occurred in the following order: imidazolium (15-20 cm(-1)) > Ntau-protonated (5-10 cm(-1)) > Npi-protonated approximately imidazolate ( approximately 0 cm(-1)). On the basis of these findings together with the Fermi resonance peaks at >2500 cm(-1) as a marker of N-H hydrogen-bonding, we concluded that the histidine residue in the S(2)/S(1) spectrum is protonated at the Npi site and that this Npi-H is hydrogen bonded. This histidine side chain probably ligated the redox-active Mn ion at the Ntau site, and thus, oxidation of the Mn cluster upon S(2) formation perturbed the histidine vibrations, causing this histidine to appear in the S(2)/S(1) difference spectrum.

The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin.

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.

Light-induced FTIR difference spectroscopy as a powerful tool toward understanding the molecular mechanism of photosynthetic oxygen evolution

The molecular mechanism of photosynthetic oxygen evolution remains a mystery in photosynthesis research. Although recent X-ray crystallographic studies of the photosystem II core complex at 3.0–3.5 Å resolutions have revealed the structure of the oxygen-evolving center (OEC), with approximate positions of the Mn and Ca ions and the amino acid ligands, elucidation of its detailed structure and the reactions during the S-state cycle awaits further spectroscopic investigations. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was first applied to the OEC in 1992 as detection of its structural changes upon the S1→S2 transition, and spectra during the S-state cycle induced by consecutive flashes were reported in 2001. These FTIR spectra provide extensive structural information on the amino acid side groups, polypeptide chains, metal core, and water molecules, which constitute the OEC and are involved in its reaction. FTIR spectroscopy is thus becoming a powerful tool in investigating the reaction mechanism of photosynthetic oxygen evolution. In this mini-review, the measurement method of light-induced FTIR spectra of OEC is introduced and the results obtained thus far using this technique are summarized.

Factors controlling the efficiency of energy transfer from carotenoids to bacteriochlorophyll in purple photosynthetic bacteria

Abstract Efficiencies of energy transfer from carotenoids to bacteriochlorophyll in purple photosynthetic bacteria have been studied with chromatophores, isolated pigment-protein complexes, and pigment-protein complexes reconstituted with a variety of carotenoids. Based on the efficiencies of energy transfer and the chemical structure of major carotenoids, photosynthetic bacteria used in this study are classified into two groups. (1) Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodocyclus gelatinosus show relatively high efficiencies (>70%) and contain spheroidene-series carotenoids which have nine or ten conjugated C=C bonds. (2) Rhodopseudomonas palustris, Rhodospirillum rubrum, and Chromatium vinosum show relatively low efficiencies (

Monitoring Water Reactions during the S-State Cycle of the Photosynthetic Water-Oxidizing Center : Detection of the DOD Bending Vibrations by Means of Fourier Transform Infrared Spectroscopy

Photosynthetic water oxidation takes place in the water-oxidizing center (WOC) of photosystem II (PSII). To clarify the mechanism of water oxidation, detecting water molecules in the WOC and monitoring their reactions at the molecular level are essential. In this study, we have for the first time detected the DOD bending vibrations of functional D 2O molecules during the S-state cycle of the WOC by means of Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra upon S-state transitions were measured using the PSII core complexes from Thermosynechococcus elongatus moderately deuterated with D 2 (16)O and D 2 (18)O. D 2 (16)O-minus-D 2 (18)O double difference spectra at individual S-state transitions exhibited six to eight peaks arising from the D (16)OD/D (18)OD bending vibrations in the 1250-1150 cm (-1) region. This observation indicates that at least two water molecules, not in any deprotonated forms, participate in the reaction at each S-state transition throughout the cycle. Most of the peaks exhibited clear counter peaks with opposite signs at different transitions, reflecting a series of reactions of water molecules at the catalytic site. In contrast, negative bands at approximately 1240 cm (-1) in the S 2 --> S 3, S 3 --> S 0, and possibly S 0 --> S 1 transitions, for which no clear counter peaks were found in other transitions, can be interpreted as insertion of substrate water into the WOC from a water cluster in the proteins. The characteristics of the weakly D-bonded OD stretching bands were consistent with the insertion of substrate from internal water molecules in the S 2 --> S 3 and S 3 --> S 0 transitions. The results of this study show that FTIR detection of the DOD bending vibrations is a powerful method for investigating the molecular mechanism of photosynthetic water oxidation as well as other enzymatic reactions involving functional water molecules.