Ryouta Takahashi

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA3 gene compared to that deduced from the psbA1 gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA1 and psbA2 genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of PheoD1 is increased by 17 mV from -522 mV in PsbA1-PSII to -505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S2QA-* charge recombination and the C identical with N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the QB pocket. The temperature dependences of the S2QA-* charge recombination show that the strength of the H-bond to PheoD1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of QA (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P680+* and YZ is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P680/P680+* couple (and hence that of 1P680*/P680+*) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.

Correlation between the hydrogen-bond structures and the C=O stretching frequencies of carboxylic acids as studied by density functional theory calculations: theoretical basis for interpretation of infrared bands of carboxylic groups in proteins.

Carboxylic groups (COOH) of Asp and Glu side chains often function as key components in enzymatic reactions, and identifying their H-bond structures in the active sites is essential for understanding the reaction mechanisms. In this study, the correlation between the H-bond structures and the C=O stretching (nuC=O) frequencies of COOH groups was studied using density functional theory calculations. The nuC=O frequencies and their shifts upon OH deuteration were calculated for model complexes of acetic acid and propionic acid H bonded at different sites with various compounds. Calculation results together with some experimental data showed that, upon direct H bonding at the C=O group, the nuC=O frequencies downshift from the free value (1770-1780 cm(-1) in an Ar matrix) to 1745-1760 cm(-1), while H bonding at the OH hydrogen induce even larger downshifts to provide the frequencies at 1720-1745 cm(-1). In contrast, when the COH oxygen is H-bonded, the nuC=O frequencies upshift to 1785-1800 cm(-1). In double and multiple H-bond forms, H-bonding effects at individual sites are basically additive, and complexes in which the C=O and the OH hydrogen are simultaneously H bonded exhibit significantly low nuC=O frequencies at 1725-1700 cm(-1), while complexes H bonded at the oxygen of the COH in addition to either at the C=O or the OH hydrogen exhibit medium frequencies of 1740-1765 cm(-1). The nuC=O frequencies linearly correlate with the C=O lengths, which are changed by H bonding at different sites. Upon OH deuteration, all the complexes showed nuC=O downshifts mostly by approximately 10 cm(-1) and in some cases as large as approximately 20 cm(-1), and hence deuteration-induced downshifts can be a good indicator, irrespective of H-bond forms, for assignments of the nuC=O bands of carboxylic groups. The results in this study provide the criteria for determining the H-bond structures of Asp and Glu side chains in proteins using their nuC=O bands in Fourier transform infrared spectra.

Differences in the Effects of Solution Additives on Heat‐ and Refolding‐Induced Aggregation

Although a number of low‐molecular‐weight additives have been developed to suppress protein aggregation, it is unclear whether these aggregation suppressors affect various aggregation processes in the same manner. In this study, we evaluated the differences in the effect of solution additives on heat‐ and refolding‐induced aggregation in the presence of guanidine (Gdn), arginine (Arg), and spermidine (Spd), and the comparable analysis showed the following differences: (i) Gdn did not suppress thermal aggregation but increased the yield of oxidative refolding. (ii) Spd showed the highest effect for heat‐induced aggregation suppression among tested compounds, although it promoted aggregation in oxidative refolding. (iii) Arg was effective for both aggregation processes. Lysozyme solubility assay and thermal unfolding experiment showed that Spd was preferentially excluded from native lysozyme and Arg and Gdn solubilized the model state of intermediates during oxidative refolding. This preference of additives to protein surfaces is the cause of the different effect on aggregation suppression.

Structures and Binding Sites of Phenolic Herbicides in the QB Pocket of Photosystem II

Herbicides targeting photosystem II (PSII) block the electron transfer beyond Q(A) by binding to the Q(B) site. Upon binding, the redox potential of Q(A) shifts differently depending on the types of herbicides. In this study, we have investigated the structures, interactions, and locations of phenolic herbicides in the Q(B) site to clarify the molecular mechanism of the Q(A) potential shifts by herbicides. Fourier transform infrared (FTIR) difference spectra upon photoreduction of the preoxidized non-heme iron (Fe(2+)/Fe(3+) difference) were measured with PSII membranes in the presence of bromoxynil or ioxynil. The CN and CO stretching vibrations of these phenolic herbicides were identified at 2215-2200 and 1516-1505 cm(-1), respectively, in the Fe(2+)/Fe(3+) difference spectra. Comparison with the spectra of bromoxynil in ethanol solutions along with density functional theory analysis strongly suggests that the phenolic herbicides take a deprotonated form in the binding pocket. In addition, the CN stretching, NH bending, and NH stretching vibrations of a His side chain, which were found at 1109-1101, 1187-1185, and 3000-2500 cm(-1), respectively, in the Fe(2+)/Fe(3+) difference spectra, showed characteristic features in the presence of phenolic herbicides. These signals are probably attributed to D1-His215, one of the ligands to the non-heme iron. Docking calculations for herbicides to the Q(B) pocket confirmed the binding of deprotonated bromoxynil to D1-His215 at the CO group, whereas the protonated form of bromoxynil and DCMU were found to bind to the opposite side of the pocket without an interaction with D1-His215. From these results, it is proposed that a strong hydrogen bond of the phenolate CO group with D1-His215 induces the change in the hydrogen bond strength of the Q(A) CO group through the Q(A)-His-Fe-His-phenolate bridge causing the downshift of the Q(A) redox potential.

Fourier transform infrared detection of a polarizable proton trapped between photooxidized tyrosine YZ and a coupled histidine in photosystem II: relevance to the proton transfer mechanism of water oxidation.

The redox-active tyrosine YZ (D1-Tyr161) in photosystem II (PSII) functions as an immediate electron acceptor of the Mn4Ca cluster, which is the catalytic center of photosynthetic water oxidation. YZ is also located in the hydrogen bond network that connects the Mn4Ca cluster to the lumen and hence is possibly related to the proton transfer process during water oxidation. To understand the role of YZ in the water oxidation mechanism, we have studied the hydrogen bonding interactions of YZ and its photooxidized neutral radical YZ(•) together with the interaction of the coupled His residue, D1-His190, using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The YZ(•)-minus-YZ FTIR difference spectrum of Mn-depleted PSII core complexes exhibited a broad positive feature around 2800 cm(-1), which was absent in the corresponding spectrum of another redox-active tyrosine YD (D2-Tyr160). Analyses by (15)N and H/D substitutions, examination of the pH dependence, and density functional theory and quantum mechanics/molecular mechanics (QM/MM) calculations showed that this band arises from the N-H stretching vibration of the protonated cation of D1-His190 forming a charge-assisted strong hydrogen bond with YZ(•). This result provides strong evidence that the proton released from YZ upon its oxidation is trapped in D1-His190 and a positive charge remains on this His. The broad feature of the ~2800 cm(-1) band reflects a large proton polarizability in the hydrogen bond between YZ(•) and HisH(+). QM/MM calculations further showed that upon YZ oxidation the hydrogen bond network is rearranged and one water molecule moves toward D1-His190. From these data, a novel proton transfer mechanism via YZ(•)-HisH(+) is proposed, in which hopping of the polarizable proton of HisH(+) to this water triggers the transfer of the proton from substrate water to the luminal side. This proton transfer mechanism could be functional in the S2 → S3 transition, which requires proton release before electron transfer because of an excess positive charge on the Mn4Ca cluster.

Effect of charge distribution over a chlorophyll dimer on the redox potential of P680 in photosystem II as studied by density functional theory calculations.

The effect of charge distribution over a chlorophyll dimer on the redox potential of P680 in photosystem II was studied by density functional theory calculations using the P680 coordinates in the X-ray structure. From the calculated ionization potentials of the dimer and the monomeric constituents, the decrease in the redox potential by charge delocalization over the dimer was estimated to be approximately 140 mV. Such charge delocalization was previously observed in the isolated D1-D2-Cyt b 559 complexes, whereas the charge was primarily localized on P D1 in the core complexes. The calculated potential decrease of approximately 140 mV can explain the inhibition of Y Z oxidation in the former complexes and in turn implies that the charge localization on P D1 upon formation of the core complex increases the P680 potential to the level necessary for water oxidation.

Hydrogen bond interactions of the pheophytin electron acceptor and its radical anion in photosystem II as revealed by Fourier transform infrared difference spectroscopy.

The primary electron acceptor pheophytin (Pheo(D1)) plays a crucial role in regulation of forward and backward electron transfer in photosystem II (PSII). It is known that some cyanobacteria control the Pheo(D1) potential in high-light acclimation by exchanging the D1 proteins from different copies of the psbA genes. To clarify the mechanism of the potential control of Pheo(D1), we studied the hydrogen bond interactions of Pheo(D1) in the neutral and anionic states using light-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra of Pheo(D1) upon its photoreduction were obtained using three different PSII preparations, PSII core complexes from Thermosynechococcus elongatus possessing PsbA1 as a D1 subunit (PSII-PsbA1), those with PsbA3 (PSII-PsbA3), and PSII membranes from spinach. The D1-Gln130 side chain, which is hydrogen bonded to the 13(1)-keto C=O group of Pheo(D1) in PSII-PsbA1, is replaced by Glu in PSII-PsbA3 and spinach PSII. The spectrum of PSII-PsbA1 exhibited 13(1)-keto C=O bands at 1682 and 1605 cm(-1) in neutral Pheo(D1) and its anion, respectively, while the corresponding bands were observed at frequencies lower by 1-3 and 18-19 cm(-1), respectively, in the latter two preparations. This larger frequency shift in Pheo(D1)(-) than Pheo(D1) by the change of the hydrogen bond donor was well reproduced by density functional theory (DFT) calculations for the Pheo models hydrogen bonded with acetamide and acetic acid. The DFT calculations also exhibited a higher redox potential for Pheo reduction in the model with acetic acid than that with acetamide, consistent with previous observations for the D1-Gln130Glu mutant of Synechocystis. It is thus concluded that a stronger hydrogen bond effect on the Pheo(-) anion than the neutral Pheo causes the shift in the redox potential, which is utilized in the photoprotection mechanism of PSII.

Structural coupling of a tyrosine side chain with the non-heme iron center in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy.

The non-heme iron is located between the quinone electron acceptors, QA and QB, in photosystem II (PSII), and together with its bicarbonate ligand, it regulates the electron and proton transfer reactions of quinone acceptors. In this study, we have investigated the structural coupling of a nearby Tyr residue with the non-heme iron center using Fourier transform infrared (FTIR) spectroscopy. Light-induced Fe2+/Fe3+ FTIR difference spectra of PSII core complexes from unlabeled and [4-13C]Tyr-labeled Thermosynechococcus elongatus revealed that the CO stretching (nuCO) bands of a Tyr side chain are located at 1253 and 1241 cm(-1) in the Fe2+ and Fe3+ states, respectively. Upon deuteration, both nuCO bands were upshifted by 11-12 cm(-1). Taking into account the criteria for determining the hydrogen bond structure of a Tyr side chain from infrared bands reported previously [Takahashi, R., and Noguchi, T. (2007) J. Phys. Chem. B 111, 13833-13844] and the results of DFT calculations of model complexes of p-cresol hydrogen-bonded with bicarbonate, we interpreted the observed nuCO bands and their deuteration effects as indicating that one Tyr side chain with a hydrogen bond donor-acceptor form is strongly coupled to the non-heme iron. From the X-ray structures of PSII core complexes, it is proposed that either D1-Y246 or D2-Y244 provides a hydrogen bond to the oxygen of the bicarbonate ligand but the other Tyr does not directly interact with bicarbonate. The Tyr residue coupled to the non-heme iron may play a key role in the regulatory function of the iron-bicarbonate center by stabilizing the bicarbonate ligand and forming a rigid hydrogen bond network around the non-heme ion.

Structural Coupling of Water Molecules with YD in Photosystem II as Revealed by FTIR Spectroscopy

The redox-active tyrosine YD (D2- Tyr160) in photosystem II (PSII) serves as an accessory electron donor to P680. It undergoes proton-coupled electron transfer; when oxidized, a proton is released and a neutral radical YD • is formed. The H-bond network around YD must play an important role in this YD reaction. In this study, we have detected water molecules structurally coupled to YD by means of light-induced Fourier transform infrared (FTIR) spectroscopy. Light-induced YD •/YD FTIR difference spectra were obtained using moderately hydrated (or deuterated) films of PSII core complexes from Thermosynechococcus elongatus at 10°C. The YD •/YD spectrum of a PSII film hydrated with unlabeled H2O showed several peaks at 3,655–3,560 cm−1 in the weakly H-bonded OH stretching region. All the peaks in this region downshifted by 11–12 cm−1 upon H2 18O substitution, while they disappeared in a deuterated film. Thus, these peaks were definitely assigned to the weakly H-bonded OH vibrations of water molecules. These results indicate that at least two water molecules are structurally coupled to YD and their H-bond interactions are perturbed upon YD oxidation. These water molecules are probably involved in a H-bond network around YD; thus, they might play a crucial role in the proton release reaction of YD.