Cryogenic Trapping and Isotope Editing Identify a Protonated Water Cluster as an Intermediate in the Photosynthetic Oxygen-Evolving Reaction

Internal water is known to play a catalytic role in several enzymes. In photosystem II (PSII), water is the substrate. To oxidize water, the PSII Mn₄CaO₅ cluster or oxygen evolving center (OEC) cycles through five oxidation states, termed S_<i>n</i> states. As reaction products, molecular oxygen is released, and protons are transferred through a ∼25 Å hydrogen-bonded network from the OEC to the thylakoid lumen. Previously, it was reported that a broad infrared band at 2880 cm^–1 is produced during the S₁-to-S₂ transition and accompanies flash-induced, S state cycling at pH 7.5. Here, we report that when the S₂ state is trapped by continuous illumination under cryogenic conditions (190 K), an analogous 2740/2900 cm^–1 band is observed. The frequency depended on the sodium chloride concentration. This band is unambiguously assigned to a normal mode of water by D₂¹⁶O and H₂¹⁸O solvent exchange. Its large, apparent H₂¹⁸O isotope shift, ammonia sensitivity, frequency, and intensity support assignment to a stretching vibration of a hydronium cation, H₃O⁺, in a small, protonated internal water cluster, <i>n</i>H₂O­(H₃O⁺). Water OH stretching bands, which may be derived from the hydration shell of the hydronium ion, are also identified. Using the 2740 cm^–1 infrared marker, the results of calcium depletion and strontium reconstitution on the protonated water cluster are found to be pH dependent. This change is attributed to protonation of an amino acid side chain and a possible change in <i>n</i>H₂O­(H₃O)⁺ localization in the hydrogen-bonding network. These results are consistent with an internal water cluster functioning as a proton acceptor and an intermediate during the S₁-to-S₂ transition. Our experiments demonstrate the utility of this infrared signal as a novel functional probe in PSII

Tracking Reactive Water and Hydrogen-Bonding Networks in Photosynthetic Oxygen Evolution

ConspectusIn oxygenic photosynthesis, photosystem II (PSII) converts water to molecular oxygen through four photodriven oxidation events at a Mn₄CaO₅ cluster. A tyrosine, YZ (Y161 in the D1 polypeptide), transfers oxidizing equivalents from an oxidized, primary chlorophyll donor to the metal center. Calcium or its analogue, strontium, is required for activity. The Mn₄CaO₅ cluster and YZ are predicted to be hydrogen bonded in a water-containing network, which involves amide carbonyl groups, amino acid side chains, and water. This hydrogen-bonded network includes amino acid residues in intrinsic and extrinsic subunits. One of the extrinsic subunits, PsbO, is intrinsically disordered. This extensive (35 Å) network may be essential in facilitating proton release from substrate water. While it is known that some proteins employ internal water molecules to catalyze reactions, there are relatively few methods that can be used to study the role of water. In this Account, we review spectroscopic evidence from our group supporting the conclusion that the PSII hydrogen-bonding network is dynamic and that water in the network plays a direct role in catalysis. Two approaches, transient electron paramagnetic resonance (EPR) and reaction-induced FT-IR (RIFT-IR) spectroscopies, were used. The EPR experiments focused on the decay kinetics of YZ• via recombination at 190 K and the solvent isotope, pH, and calcium dependence of these kinetics. The RIFT-IR experiments focused on shifts in amide carbonyl frequencies, induced by photo-oxidation of the metal cluster, and on the isotope-based assignment of bands to internal, small protonated water clusters at 190, 263, and 283 K. To conduct these experiments, PSII was prepared in selected steps along the catalytic pathway, the S_<i>n</i> state cycle (<i>n</i> = 0–4). This cycle ultimately generates oxygen. In the EPR studies, S-state dependent changes were observed in the YZ• lifetime and in its solvent isotope effect. The YZ• lifetime depended on the presence of calcium at pH 7.5, but not at pH 6.0, suggesting a two-donor model for PCET. At pH 6.0 or 7.5, barium and ammonia both slowed the rate of YZ• recombination, consistent with disruption of the hydrogen-bonding network. In the RIFT-IR studies of the S state transitions, infrared bands associated with the transient protonation and deprotonation of internal waters were identified by D₂O and H₂¹⁸O labeling. The infrared bands of these protonated water clusters, W_<i>n</i>⁺ (or <i>n</i>H₂O­(H₃O)⁺, <i>n</i> = 5–6), exhibited flash dependence and were produced during the S₁ to S₂ and S₃ to S₀ transitions. Calcium dependence was observed at pH 7.5, but not at pH 6.0. S-state induced shifts were observed in amide CO frequencies during the S₁ to S₂ transition and attributed to alterations in hydrogen bonding, based on ammonia sensitivity. In addition, isotope editing of the extrinsic subunit, PsbO, established that amide vibrational bands of this lumenal subunit respond to the S state transitions and that PsbO is a structural template for the reaction center. Taken together, these spectroscopic results support the hypothesis that proton transfer networks, extending from YZ to PsbO, play a functional and dynamic role in photosynthetic oxygen evolution

Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with colorectal cancer outcome

<p>Single nucleotide polymorphisms (SNPs) in the displacement loop (D-Loop) of mitochondrial DNA (mtDNA) has been identified for their association with the risk and outcome in many cancers. We have identified risk associated D-loop SNPs for colorectal cancer previously, in the present study, we evaluate their prognostic value for postoperative survival of colorectal cancer (CRC). The minor haplotype of nucleotides 16290T and frequent haplotype of nucleotide 16298T in the hypervariable segment 1 (HV1) region of the D-loop were identified for their association with high survival rate of CRC. After adjusted with COX proportional hazard model, the nucleotide site of 16290 was identified as independent predictor for CRC (RR, 0.379; 95% CI, 0.171–0.839; <i>p</i> = 0.017). In conclusion, SNPs in the mtDNA D-Loop were found to be valuable markers for colorectal cancer outcome evaluation.</p

Univariate analysis of HBV polymerase gene mutations associated with postoperative survival in patients with HBV-HCC.

<p>Univariate analysis of HBV polymerase gene mutations associated with postoperative survival in patients with HBV-HCC.</p

Mutations in hepatitis B virus polymerase are associated with the postoperative survival of hepatocellular carcinoma patients

<div><p>Proofreading deficiencies of hepatitis B virus polymerase result in frequent DNA mutations in the hepatitis B virus genome. Here, we performed sequencing analysis of the hepatitis B virus polymerase gene to assess its association with the postoperative survival in 92 patients with HBV-related hepatocellular carcinoma by using the Kaplan–Meier method. The 2525, 2733, 2738, 2768, 2946, 3063, 3066, 3109, 31, 529, 735, 939, 1078, 1137, 1383, 1461, 1485, 1544, and 1613 mutation sites were identified as being associated with HCC outcomes by the log-rank test. After adjusting for clinical characteristics by using the Cox hazard model, site 31 (relative risk, 8.929; 95% confidence interval, 3.433–23.22; <i>P</i> = 0.000) in the spacer domain and sites 529 (relative risk, 5.656; 95% confidence interval, 1.599–19.999; <i>P</i> = 0.007) and 1078 (relative risk, 3.442; 95% confidence interval, 1.070–11.068; <i>P</i> = 0.038) in the reverse transcriptase domain of hepatitis B virus polymerase were identified as independent predictors of postoperative survival in hepatitis B virus related hepatocellular carcinoma. The mutations at the 31 (Ser314Pro), 529 (Asp480Asn), and 1078 (Ser663Ala) sites all resulted in amino acid changes in hepatitis B virus polymerase and were associated with shortened life-span. The 31 and 529 sites were located in the overlapping region for the PreS and S genes but did not induce amino acid substitution in these two regions. Our finding of the correlation between hepatitis B virus DNA polymerase mutations and hepatocellular carcinoma survival will help identify the patients subgroup with poor prognosis, and help the clinicians to refine the therapeutic decision individualized.</p></div

Distribution of mutation frequency in the HBV polymerase region.

<p>The square indicates the mutations associated with survival in HBV-HCC, as determined by univariate analysis using the log-rank test; the rhombus indicates mutations that are not associated with HBV-HCC.</p

Multivariate analysis of the clinical factors associated with postoperative survival in HBV-HCC patients.

<p>Multivariate analysis of the clinical factors associated with postoperative survival in HBV-HCC patients.</p

Univariate and multivariate analysis of the clinical factors associated with postoperative survival in HBV-HCC patients.

<p>Univariate and multivariate analysis of the clinical factors associated with postoperative survival in HBV-HCC patients.</p

Schema of the polymerase region and overlapping preS, S, and X regions.

<p>Schema of the polymerase region and overlapping preS, S, and X regions.</p