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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2021)

Our current understanding of biological C-H bond activation in heme enzymes points to an important role of basic ferryl intermediates in how Nature supports this chemistry. Among heme proteins, only the thiolate-ligated enzymes have been demonstrated to react with unactivated C-H bonds. Strong electron donation from the proximal thiolate ligand results in a basic Compound II species, an iron(IV) hydroxide. This is important for balancing the reduction potential of Compound I and avoiding deleterious autoxidation of the protein superstructure. However, recent neutron crystallography experiments point to a basic ferryl species present in a histidine-ligated heme enzyme as well, which have been previously shown to only support unprotonated oxos. Our work demonstrates that ascorbate peroxidase, the histidine-ligated heme enzyme, really has an unprotonated iron(IV) oxo Compound II species (APX-II). We further examine the iron(III) hydroxide state of ascorbate peroxidase in order to better understand the reported crystal structure and what it represents. We conclude with a vibrational spectroscopic method to observe the Fe-O bond in APX-II and assign it to an Fe(IV) to a doubly bound oxygen atom, split between multiple vibrational modes resulting in apparent stretching frequencies of 770 and 732 cm-1 , or 1.67 and 1.69 Å, respectively. This is well within range of what is expected for an iron(IV) oxo

    Cover page: Understanding Ferryl Basicity And Proximal Histidine Ligation
    • Thesis
    • Peer Reviewed
    UC Irvine Electronic Theses and Dissertations (2023)

    Although the activation of inert C–H bonds by metal-oxo complexes has been widely studied, important questions remain, particularly regarding the roles of oxygen spin population (i.e., unpaired electrons on the oxo ligand), the antiferromagnetic coupling and low lying excited states in facilitating C–H bond cleavage. In order to investigate these factors, we have utilized experimental and computational methods. Pulse EPR experiments were performed on isotopically labelled heme enzyme compound I intermediates to measure spin populations. Semiempirical modeling was utilized to relate spectroscopically obtained parameters to information about the excited states.

      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2019)
      The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein-protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres-including a penta-His-coordinated non-haem Fe site-and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.
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      Cover page: An efficient, step-economical strategy for the design of functional metalloproteins.
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      The oxidation of substrates via the cleavage of thermodynamically strong C-H bonds is an essential part of mammalian metabolism. These reactions are predominantly carried out by enzymes that produce high-valent metal-oxido species, which are directly responsible for cleaving the C-H bonds. While much is known about the identity of these transient intermediates, the mechanistic factors that enable metal-oxido species to accomplish such difficult reactions are still incomplete. For synthetic metal-oxido species, C-H bond cleavage is often mechanistically described as synchronous, proton-coupled electron transfer (PCET). However, data have emerged that suggest that the basicity of the M-oxido unit is the key determinant in achieving enzymatic function, thus requiring alternative mechanisms whereby proton transfer (PT) has a more dominant role than electron transfer (ET). To bridge this knowledge gap, the reactivity of a monomeric MnIV-oxido complex with a series of external substrates was studied, resulting in a spread of over 104 in their second-order rate constants that tracked with the acidity of the C-H bonds. Mechanisms that included either synchronous PCET or rate-limiting PT, followed by ET, did not explain our results, which led to a proposed PCET mechanism with asynchronous transition states that are dominated by PT. To support this premise, we report a semiempirical free energy analysis that can predict the relative contributions of PT and ET for a given set of substrates. These findings underscore why the basicity of M-oxido units needs to be considered in C-H functionalization.
        Cover page: Semiempirical method for examining asynchronicity in metal–oxido-mediated C–H bond activation
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2016)
        High-valent Fe-OH species are often invoked as key intermediates but have only been observed in Compound II of cytochrome P450s. To further address the properties of non-heme FeIV-OH complexes, we demonstrate the reversible protonation of a synthetic FeIV-oxo species containing a tris-urea tripodal ligand. The same protonated FeIV-oxo species can be prepared via oxidation, suggesting that a putative FeV-oxo species was initially generated. Computational, Mössbauer, XAS, and NRVS studies indicate that protonation of the FeIV-oxo complex most likely occurs on the tripodal ligand, which undergoes a structural change that results in the formation of a new intramolecular H-bond with the oxido ligand that aids in stabilizing the protonated adduct. We suggest that similar protonated high-valent Fe-oxo species may occur in the active sites of proteins. This finding further argues for caution when assigning unverified high-valent Fe-OH species to mechanisms.
          Cover page: Reactivity of an FeIV-Oxo Complex with Protons and Oxidants
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2020)
          High-valent nonheme FeIV-oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal-oxido complexes. To address this knowledge gap, we introduced a series of FeIV-oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat]3-, which contains phosphinic amido groups. An important structural aspect of [FeIVpoat(O)]- is the inclusion of an auxiliary site capable of binding a Lewis acid (LAII); we used this unique feature to further modulate the electrostatic environment around the Fe-oxido unit. Experimentally, studies confirmed that H-bonds and LAII s can interact directly with the oxido ligand in FeIV-oxido complexes, which weakens the Fe═O bond and has an impact on the electronic structure. We found that relatively large vibrational changes in the Fe-oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.
            Cover page: Effects of Noncovalent Interactions on High-Spin Fe(IV)–Oxido Complexes
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2020)
            An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.
              Cover page: Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases
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