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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2020)

Oxygenic photosynthesis produces nearly all the O2 on Earth and sustains nearly all of its biomass. This process is catalyzed by Photosystem II (PSII), a large, transmembrane protein embedded in the thylakoid membranes of plants, algae, and cyanobacteria. PSII is catalyzed by a Mn4CaO5 cluster. As light energy is absorbed by the reaction centers of PSII, the Mn4CaO5 cluster accumulates oxidizing equivalents through a discreet and precisely choreographed light-induced electron transfer. The light induced oxidations cause the catalytic cluster to cycle through five oxidation states, Sn (n=0-4) where ‘n’ refers to the number of oxidizing equivalents. After formation of the S4 state, the cluster oxidizes two water molecules, releases O2 and returns to the S0 state, the lowest oxidation state of its catalytic cycle. The oxidation of water is a thermodynamically and kinetically demanding reaction. This is managed by PSII through the careful choreography of proton and electron transfers to the Mn4CaO5 cluster throughout the catalytic cycle. The Mn4CaO5’s reactivity in each catalytic step is carefully controlled by its protein environment. Identifying key amino acid residues, determining their responsibility in the reaction, and characterizing the proton egress pathways during the individual S-state transitions are paramount in understanding the oxygen evolution mechanism.

This study interrogates and identifies the amino acid residues responsible for controlling the Mn¬4CaO5 cluster’s reactivity and determines the role of each residue through Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectroscopy is capable of characterizing the dynamic structural rearrangements during PSII’s catalytic cycle. A combination of site-directed mutagenesis (D1-V185N, D1-S169A, D1-E189G, D1-E189S, D1-E329A), isotopic substitutions, and the substitution of Sr2+ for Ca2+ in PSII’s catalytic center were used to further delineate the dominant water access and proton egress pathways that link the catalytic cluster with the thylakoid lumen, characterize the influence of specific protein residues on substrate water molecules and on the network of hydrogen bonds in these pathways, and elucidate the potential substrates and mechanisms of the S2 to S3 transition.

    Cover page: Light-Induced Fourier Transform Infrared Difference Spectroscopy Study of the Molecular Mechanism of Photosynthetic Oxygen Evolution
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2021)
    The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the 'slow exchanging substrate water (Ws)', while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the 'fast exchanging substrate water (Wf)'. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H2 16O/H2 18O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution.
      Cover page: The exchange of the fast substrate water in the S 2 state of photosystem II is limited by diffusion of bulk water through channels – implications for the water oxidation mechanism
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2018)
      Photoinduced water oxidation at the O2-evolving complex (OEC) of photosystem II (PSII) is a complex process involving a tetramanganese-calcium cluster that is surrounded by a hydrogen-bonded network of water molecules, chloride ions, and amino acid residues. Although the structure of the OEC has remained conserved over eons of evolution, significant differences in the chloride-binding characteristics exist between cyanobacteria and higher plants. An analysis of amino acid residues in and around the OEC has identified residue 87 in the D1 subunit as the only significant difference between PSII in cyanobacteria and higher plants. We substituted the D1-Asn87 residue in the cyanobacterium Synechocystis sp. PCC 6803 (wildtype) with alanine, present in higher plants, or with aspartic acid. We studied PSII core complexes purified from D1-N87A and D1-N87D variant strains to probe the function of the D1-Asn87 residue in the water-oxidation mechanism. EPR spectra of the S2 state and flash-induced FTIR spectra of both D1-N87A and D1-N87D PSII core complexes exhibited characteristics similar to those of wildtype Synechocystis PSII core complexes. However, flash-induced O2-evolution studies revealed a decreased cycling efficiency of the D1-N87D variant, whereas the cycling efficiency of the D1-N87A PSII variant was similar to that of wildtype PSII. Steady-state O2-evolution activity assays revealed that substitution of the D1 residue at position 87 with alanine perturbs the chloride-binding site in the proton-exit channel. These findings provide new insight into the role of the D1-Asn87 site in the water-oxidation mechanism and explain the difference in the chloride-binding properties of cyanobacterial and higher-plant PSII.
        Cover page: Substitution of the D1-Asn87 site in photosystem II of cyanobacteria mimics the chloride-binding characteristics of spinach photosystem II
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2017)

        Context

        - Immunomarkers with diagnostic, therapeutic, or prognostic values have been increasingly used to maximize the benefits of clinical management of patients with neoplastic diseases of the gastrointestinal tract, liver, biliary tract, and pancreas.

        Objectives

        - To review the characteristics of immunomarkers that are commonly used in surgical pathology practice for neoplasms of the gastrointestinal tract, liver, biliary tract, and pancreas, and to summarize the clinical usefulness of immunomarkers that have been discovered in recent years in these fields.

        Data sources

        - Data sources include literature review, authors' research data, and personal practice experience.

        Conclusions

        - Immunohistochemistry is an indispensable tool for the accurate diagnosis of neoplastic diseases of the gastrointestinal tract, liver, biliary tract, and pancreas. Useful immunomarkers are available to help distinguish malignant neoplasms from benign conditions, determine organ origins, and subclassify neoplasms that are morphologically and biologically heterogeneous. Specific immunomarkers are also available to help guide patient treatment and assess disease aggressiveness, which are keys to the success of personalized medicine. Pathologists will continue to play a critical role in the discovery, validation, and application of new biomarkers, which will ultimately improve patient care.
          Cover page: Practical Immunohistochemistry in Neoplastic Pathology of the Gastrointestinal Tract, Liver, Biliary Tract, and Pancreas
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