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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2010)

The Heme-Nitric oxide/OXygen binding (H-NOX) domain encompasses a family of proteins closely related (≤ 40% sequence identity) to the heme domain of soluble guanylate cyclase (sGC), the eukaryotic enzyme receptor for NO [1, 2]. sGC discriminates between NO and O2, and exclusively binds NO even in the presence of excess O2. Although some H-NOX domains have ligand binding properties that are identical to sGC, others additionally bind O2, including the atypical sGCs from Drosophila melanogaster and Caenorhabditis elegans [1, 3, 4]. The molecular basis of this ligand selectivity is not fully understood. In order to better understand the H-NOX domain, resonance Raman (RR) spectroscopy was employed in combination with isotopic substitution and site-directed mutagenesis to probe the heme pocket in Thermoanaerobacter tengcongensis (Tt H-NOX) as a model system for this family of heme proteins. Thus, the expression, purification, and RR characterization of Tt H-NOX and selected mutants were performed to elucidate the heme environmental properties that influence ligand binding.

A striking feature of the O2-bound WT Tt H-NOX crystal structure is the presence of a distorted heme molecule [5]. RR investigation of Tt H-NOX proteins containing mutations at key conserved residues, Ile-5 and Pro-115, determined that the most dramatic heme conformational changes occurred in the O2-bound forms, and that the single P115A mutation generated a significantly relaxed chromophore. Decreased RR intensities were observed for several out-of-plane modes in the 400-750 cm-1 region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal modes. These changes demonstrated that the P115A heme conformation was considerably more relaxed than WT, with increased flexibility within the protein pocket that allowed for rapid sampling of alternate conformations.

Another remarkable feature of this family is the spectroscopic observation of an unusually high ν(C-O) frequency. To elucidate the interactions responsible for this property, mutations were made in Tt H-NOX to probe the distal pocket, the conserved Tyr-Ser-Arg (YxSxR) motif, and the heme-linkage site (His-102). RR spectra of these mutants indicated that H-bonding interactions between these residues and the heme significantly affected the CO bond by increasing the back-donation of the FeII dπ electrons into the CO π* orbitals. The most significant change occurred upon disruption of the H-bonds between the YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations; one was structurally similar to WT, and the other conformer displayed ν(C-O) downshifts of up to ~70 cm-1. Based on these shifts, the most important factor contributing to the C-O stretching mode may be the neutralization of the negative charges on the propionate groups via the strictly conserved YxSxR motif.

Finally, the role of the Tt H-NOX binding pocket in stabilizing the O2 complex was investigated. Evidence of H-bonds to the bound O2 was demonstrated in the RR spectra of Trp-9 and Tyr-140 mutants; the disruption of the H-bond network in these mutations increased ν(Fe-O2) upon returning electron density to the Fe-O2 bond. In contrast, the addition of steric bulk to the H-NOX pocket decreased the ν(Fe-O2) frequency relative to the WT protein. These shifts suggested that the bulky distal residues forced the H-NOX binding pocket into a more open position that increased the distance between Tyr-140 and the O2, thereby weakening this crucial H-bond. Thus, two distinct factors influence the ν(Fe-O2) in Tt H-NOX: (i) electrostatic effects from H-bonding with Tyr-140, which increased the ν(Fe-O2) frequency upon its removal; and (ii) steric effects which decreased ν(Fe-O2) due to bond lengthening from a more loosely held O2 ligand.

In summary, this work presents a comparative analysis of the heme environmental effects in Tt H-NOX. By directly probing the interactions between the heme chromophore and the protein pocket through a combination of RR spectroscopy and site-directed mutagenesis, this study may elucidate the biochemical properties of these H-NOX domains, and bring insight into the relationship between heme structure and protein function in this family.

    Cover page: Probing the heme structure and environment of bacterial Heme-Nitric oxide/OXygen binding domains with resonance Raman spectroscopy
    • Article
    • Peer Reviewed
    LBL Publications (2015)
    Well-defined mixed-metal [CoMn3 O4 ] and [NiMn3 O4 ] cubane complexes were synthesized and used as precursors for heterogeneous oxygen evolution reaction (OER) electrocatalysts. The discrete clusters were dropcasted onto glassy carbon (GC) and indium tin oxide (ITO) electrodes, and the OER activities of the resulting films were evaluated. The catalytic surfaces were analyzed by various techniques to gain insight into the structure-function relationships of the electrocatalysts' heterometallic composition. Depending on preparation conditions, the Co-Mn oxide was found to change metal composition during catalysis, while the Ni-Mn oxides maintained the NiMn3 ratio. XAS studies provided structural insights indicating that the electrocatalysts are different from the molecular precursors, but that the original NiMn3 O4 cubane-like geometry was maintained in the absence of thermal treatment (2-Ni). In contrast, the thermally generated 3-Ni develops an oxide-like extended structure. Both 2-Ni and 3-Ni undergo structural changes upon electrolysis, but they do not convert into the same material. The observed structural motifs in these heterogeneous electrocatalysts are reminiscent of the biological oxygen-evolving complex in Photosystem II, including the MMn3 O4 cubane moiety. The reported studies demonstrate the use of discrete heterometallic oxide clusters as precursors for heterogeneous water oxidation catalysts of novel composition and the distinct behavior of two sets of mixed metal oxides.
      • Article
      • Peer Reviewed
      LBL Publications (2015)
      In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 μm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5Å, using crystals grown without the micro seeding approach, to 4.5Å using crystals generated with the new method.
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2013)
        Photosystem II supports four manganese centers through nine oxidation states from manganese(II) during assembly through to the most oxidized state before O2 formation and release. The protein-based carboxylate and imidazole ligands allow for significant changes of the coordination environment during the incorporation of hydroxido and oxido ligands upon oxidation of the metal centers. We report the synthesis and characterization of a series of tetramanganese complexes in four of the six oxidation states from MnII3MnIII to MnIII2 MnIV2 with the same ligand framework (L) by incorporating four oxido ligands. A 1,3,5-triarylbenzene framework appended with six pyridyl and three alkoxy groups was utilized along with three acetate anions to access tetramanganese complexes, Mn4O x , with x = 1, 2, 3, and 4. Alongside two previously reported complexes, four new clusters in various states were isolated and characterized by crystallography, and four were observed electrochemically, thus accessing the eight oxidation states from MnII4 to MnIIIMnIV3. This structurally related series of compounds was characterized by EXAFS, XANES, EPR, magnetism, and cyclic voltammetry. Similar to the ligands in the active site of the protein, the ancillary ligand (L) is preserved throughout the series and changes its binding mode between the low and high oxido-content clusters. Implications for the rational assembly and properties of high oxidation state metal-oxido clusters are presented.
          Cover page: Role of oxido incorporation and ligand lability in expanding redox accessibility of structurally related Mn 4 clusters
          • Article
          • Peer Reviewed
          LBL Publications (2015)
          Herein, Ca K-edge X-ray absorption spectroscopy (XAS) is developed as a means to characterize the local environment of calcium centers. The spectra for six, seven, and eight coordinate inorganic and molecular calcium complexes were analyzed and determined to be primarily influenced by the coordination environment and site symmetry at the calcium center. The experimental results are closely correlated to time-dependent density functional theory (TD-DFT) calculations of the XAS spectra. The applicability of this methodology to complex systems was investigated using structural mimics of the oxygen-evolving complex (OEC) of PSII. It was found that Ca K-edge XAS is a sensitive probe for structural changes occurring in the cubane heterometallic cluster due to Mn oxidation. Future applications to the OEC are discussed.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2013)
            L-edge spectroscopy of 3d transition metals provides important electronic structure information and has been used in many fields. However, the use of this method for studying dilute aqueous systems, such as metalloenzymes, has not been prevalent because of severe radiation damage and the lack of suitable detection systems. Here we present spectra from a dilute Mn aqueous solution using a high-transmission zone-plate spectrometer at the Linac Coherent Light Source (LCLS). The spectrometer has been optimized for discriminating the Mn L-edge signal from the overwhelming O K-edge background that arises from water and protein itself, and the ultrashort LCLS X-ray pulses can outrun X-ray induced damage. We show that the deviations of the partial-fluorescence yield-detected spectra from the true absorption can be well modeled using the state-dependence of the fluorescence yield, and discuss implications for the application of our concept to biological samples.
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2016)
              Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
                • Article
                • Peer Reviewed
                LBL Publications (2016)
                X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
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