William A Gunderson

Northwestern University, Evanston, Illinois, United States

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Publications (13)136.83 Total impact

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    ABSTRACT: Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His 429 and the proximal thiolate ligand, Cys 436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle we have used radiolytic cryoreduction combined with EPR and ENDOR spectroscopy to study and compare their characteristics for wild type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron (III) and the hyperfine couplings of the heme-pyrrole 14N, but has relatively little effect on the 1H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the S→Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In wild-type enzyme the added electron localizes on the porphyrin, generating an S = 3/2 state with the anion radical exchange-coupled to the Fe (II). In the mutant it localizes on the iron, generating a S = ½ Fe(I) state. (iii) The additional H-bond has little effect on g-values and 1H, 14N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both WT and mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant.(vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.
    No preview · Article · Jan 2016 · Biochemistry
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    ABSTRACT: Iron TAML activators of peroxides are functional catalase-peroxidase mimics. Switching from H2O2 to O2 as the primary oxidant was achieved by using a system of reverse micelles of Aerosol OT (AOT) in n-octane. Hydrophilic TAML activators are localized in the aqueous microreactors of reverse micelles where water is present in much lower abundance than in bulk water. n-Octane serves as a proximate reservoir supplying O2 to result in partial oxidation of Fe(III) to Fe(IV)-containing species, mostly the Fe(III)Fe(IV) (major) and Fe(IV)Fe(IV) (minor) dimers which coexist with the Fe(III) TAML monomeric species. The speciation depends on the pH and the degree of hydration w0, viz. the amount of water in the reverse micelles. The previously unknown Fe(III)Fe(IV) dimer has been characterized by the UV-vis, EPR and Mössbauer spectroscopies. Reactive electron donors such as NADH, Pinacyanol chloride and hydroquinone undergo the TAML-catalyzed oxidation by O2. The oxidation of NADH, studied in most detail, is much faster in "dry micelles" at the lowest degree of hydration w0 and is accelerated by light through NADH photochemistry. Dyes that are more resistant to oxidation than Pinacyanol chloride (Orange II, Safranine O) are not oxidized in the reverse micellar media. Despite the limitation of low reactivity, the new systems highlight an encouraging step in replacing TAML peroxidase-like chemistry with more attractive oxygen-activation chemistry.
    No preview · Article · Jul 2015 · Journal of the American Chemical Society
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    ABSTRACT: The ability of certain transition metals to mediate the reduction of N2 to NH3 has attracted broad interest in the biological and inorganic chemistry communities. Early transition metals such as Mo and W readily bind N2 and mediate its protonation at one or more N atoms to furnish M(NxHy) species that can be characterized and, in turn, extrude NH3. By contrast, the direct protonation of Fe-N2 species to Fe(NxHy) products that can be characterized has been elusive. Herein we show that addition of acid at low temperature to [(TPB)Fe(N2)][Na(12-crown-4)] results in a new S = 1/2 Fe species. EPR, ENDOR, Mössbauer, and EXAFS analysis, coupled with a DFT study, unequivocally assign this new species as [(TPB)Fe≡N-NH2]+, a doubly protonated hydra-zido(2-) complex featuring an Fe-to-N triple bond. This unstable species offers strong evidence that the first steps in Fe-mediated nitrogen reduction by [(TPB)Fe(N2)][Na(12-crown-4)] can proceed along a distal or 'Chatt-type' pathway. A brief discussion of whether subsequent catalytic steps may involve early or late stage cleavage of the N-N bond, as would be found in limiting distal or alternating mechanisms, respectively, is also provided.
    No preview · Article · May 2015 · Journal of the American Chemical Society
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    ABSTRACT: Proton exchange within the MH2 moiety of (TPB)Co(H-2) (CoH2; TPB = B(o-C(6)H(4)PiPr(2))(3)) by 2-fold rotation about the MH2 axis is probed through EPR/ENDOR studies and a neutron diffraction crystal structure. This complex is compared with previously studied (SiP3iPr)Fe(H-2) (FeH2) (SiP3iPr = [Si(o-(C6H4PPr2)-Pr-i)(3)]). The g-values for CoH2 and FeH2 show that both have the JahnTeller (JT)-active E-2 ground state (idealized C-3 symmetry) with doubly degenerate frontier orbitals, (e)(3) = [|m(L) +/- 2>](3) = [x(2) y(2), xy](3), but with stronger linear vibronic coupling for CoH2. The observation of H-1 ENDOR signals from the CoHD complex, 2H signals from the CoD2/HD complexes, but no H-1 signals from the CoH2 complex establishes that H-2 undergoes proton exchange at 2 K through rotation around the CoH2 axis, which introduces a quantum-statistical (Pauli-principle) requirement that the overall nuclear wave function be antisymmetric to exchange of identical protons (I = 1/2; Fermions), symmetric for identical deuterons (I = 1; Bosons). Analysis of the 1-D rotor problem indicates that CoH2 exhibits rotor-like behavior in solution because the underlying C3 molecular symmetry combined with H-2 exchange creates a dominant 6-fold barrier to H-2 rotation. FeH2 instead shows H-2 localization at 2 K because a dominant 2-fold barrier is introduced by strong Fe(3d)-> H-2(sigma*) pi-backbonding that becomes dependent on the H-2 orientation through quadratic JT distortion. ENDOR sensitively probes bonding along the L2ME axis (E = Si for FeH2; E = B for CoH2). Notably, the isotropic H-1/H-2 hyperfine coupling to the diatomic of CoH2 is nearly 4-fold smaller than for FeH2.
    No preview · Article · Sep 2014 · Journal of the American Chemical Society
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    ABSTRACT: Proton exchange within the M–H_2 moiety of (TPB)Co(H_2) (Co–H_2; TPB = B(o-C_6H_4PiPr_2)_3) by 2-fold rotation about the M–H_2 axis is probed through EPR/ENDOR studies and a neutron diffraction crystal structure. This complex is compared with previously studied (SiP^(iPr)_3)Fe(H_2) (Fe–H_2) (SiP^(iPr)_3 = [Si(o-C_6H_4PiPr_2)_3]). The g-values for Co–H_2 and Fe–H_2 show that both have the Jahn–Teller (JT)-active ^2E ground state (idealized C_3 symmetry) with doubly degenerate frontier orbitals, (e)^3 = [|m_L ± 2>]^3 = [x^2 – y^2, xy]^3, but with stronger linear vibronic coupling for Co–H_2. The observation of ^1H ENDOR signals from the Co–HD complex, ^2H signals from the Co–D_2/HD complexes, but no ^1H signals from the Co–H_2 complex establishes that H_2 undergoes proton exchange at 2 K through rotation around the Co–H_2 axis, which introduces a quantum-statistical (Pauli-principle) requirement that the overall nuclear wave function be antisymmetric to exchange of identical protons (I = 1/2; Fermions), symmetric for identical deuterons (I = 1; Bosons). Analysis of the 1-D rotor problem indicates that Co–H_2 exhibits rotor-like behavior in solution because the underlying C_3 molecular symmetry combined with H_2 exchange creates a dominant 6-fold barrier to H_2 rotation. Fe–H_2 instead shows H_2 localization at 2 K because a dominant 2-fold barrier is introduced by strong Fe(3d)→ H_2(σ^*) π-backbonding that becomes dependent on the H_2 orientation through quadratic JT distortion. ENDOR sensitively probes bonding along the L_2–M–E axis (E = Si for Fe–H_2; E = B for Co–H_2). Notably, the isotropic ^1H/^2H hyperfine coupling to the diatomic of Co–H_2 is nearly 4-fold smaller than for Fe–H_2.
    No preview · Article · Sep 2014 · Journal of the American Chemical Society
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    ABSTRACT: Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. As a copper-containing enzyme, pMMO has been investigated extensively by electron paramagnetic resonance (EPR) spectroscopy, but the presence of multiple copper centers has precluded correlation of EPR signals with the crystallographically identified monocopper and dicopper centers. A soluble recombinant fragment of the pmoB subunit of pMMO, spmoB, like pMMO itself, contains two distinct copper centers and exhibits methane oxidation activity. The spmoB protein, spmoB variants designed to dis-rupt one or the other or both copper centers, as well as native pMMO have been investigated by EPR, ENDOR, and ESEEM spectroscopies in combination with metal content analysis. The data are remarkably similar for spmoB and pMMO, validating the use of spmoB as a model system. The results indicate that one EPR-active Cu(II) ion is present per pMMO, and that it is associated with the active-site dicopper center in the form of a valence localized Cu(I)Cu(II) pair; the Cu(II), however, is scrambled between the two locations within the dicopper site. The monocopper site observed in the crystal structures of pMMO can be assigned as Cu(I). (14)N ENDOR and ESEEM data are most consistent with one of these dicopper-site signals involving coordination of the Cu(II) ion by residues His137 and His139, the other with Cu(II) coordinated by His33 and the N-terminal amino group. (1)H ENDOR measurements indicate there is no aqua (HxO) ligand bound to the Cu(II), either terminally or as a bridge to Cu(I).
    Preview · Article · Jul 2014 · Journal of the American Chemical Society
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    ABSTRACT: A discrete nanocage of core-shell design, in which carboxylic acid groups were tethered to the core and silanol to the shell interior, was found to react with Co2(CO)8 to form and stabilize a Co(I)-CO species. The singular CO stretching band of this new Co species at 1958 cm(-1) and its magnetic susceptibility were consistent with Co(I) compounds. When exposed to O2, it transformed from an EPR inactive to an EPR active species indicative of oxidation of Co(I) to Co(II) with the formation of H2O2. It could be oxidized also by organoazide or water. Its residing in the nanocage interior was confirmed by size selectivity in the oxidation process and the fact that the entrapped Co species could not be accessed by an electrode.
    Full-text · Article · Mar 2014 · Journal of the American Chemical Society
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    ABSTRACT: The amyloid-β (Aβ) protein forms fibrils and higher-order plaque aggegrates in Alzheimer's disease (AD) brain. The copper ion, Cu(2+), is found at high concentrations in plaques, but its role in AD etiology is unclear. We use high-resolution pulsed electron paramagnetic resonance spectroscopy to characterize the coordination structure of Cu(2+) in the fibrillar form of full-length Aβ(1-40). The results reveal a bis-cis-histidine (His) equatorial Cu(2+) coordination geometry and participation of all three N-terminal His residues in Cu(2+) binding. A model is proposed in which Cu(2+)-His6/His13 and Cu(2+)-His6/His14 sites alternate along the fibril axis on opposite sides of the β-sheet fibril structure. The local intra-β-strand coordination structure is not conducive to Cu(2+)/Cu(+) redox-linked coordination changes, and the global arrangement of Cu sites precludes facile multielectron and bridged-metal site reactivity. This indicates that the fibrillar form of Aβ suppresses Cu redox cycling and reactive oxygen species production. The configuration suggests application of Cu(2+)-Aβ fibrils as an amyloid architecture for switchable electron charge/spin coupling and redox reactivity.
    No preview · Article · Oct 2012 · Journal of the American Chemical Society
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    ABSTRACT: Extradiol catecholic dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O2. These enzymes are important in nature for the recovery of large amounts of carbon from aromatic compounds. The catalytic site contains either Fe or Mn coordinated by a facial triad of two His and one Glu or Asp residues. Previous studies have shown that Fe(II) and Mn(II) can be interchanged in enzymes from different organisms to catalyze similar substrate reactions. In combination, quantitative electron paramagnetic resonance spectroscopy and rapid freeze-quench experiments allow us to follow the concentrations of four different Mn species, including key metal intermediates in the catalytic cycle, as the enzyme turns over its natural substrate. Two intermediates are observed: a Mn(III)-radical species which is either Mn-superoxide or Mn-substrate radical, and a unique Mn(II) species which is involved in the rate-limiting step of the cycle and may be Mn-alkylperoxo.
    Full-text · Article · Nov 2008 · Journal of the American Chemical Society
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    ABSTRACT: The binding and activation of dioxygen by transition metal complexes is a fundamentally and practically important process in chemistry. Often the initial steps involve formation of peroxometal species that is difficult to observe because of their inherent reactivity. The interaction of dioxygen with a manganese(II) complex (1) of bis[(N'-tert-butylurealy)-N-ethyl]-(6-pivalamido-2-pyridylmethyl)amine was investigated, leading to the detection of a new intermediate that is a peroxomanganese(III) complex (2). This complex is high-spin (S = 2) with a g value of 8.2 and D = -2.0(5) as determined by parallel-mode electron paramagnetic resonance spectroscopy. The coordination of a peroxo ligand was established using Fourier transform infrared spectroscopy that reveals a new signal at 885 cm-1 for 2 when formed from 16O2-this band shifts to 837 cm-1 when 18O2 is used in the preparation. Moreover, electrospray ionization mass spectra contain a strong ion at an m/z of 576.2703 for the 16O-isotopomer that shifts to 580.2794 in the 18O-isotopomer. Complex 2 also is capable of oxidatively deformylating aldehydes, which is a known reaction of peroxometal complexes. The similarities of 2 to the peroxo intermediates in cytochrome P450 are noted.
    Full-text · Article · Aug 2008 · Journal of the American Chemical Society
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    Misha V Golynskiy · William A Gunderson · Michael P Hendrich · Seth M Cohen
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    ABSTRACT: Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available. Herein, the metal-binding affinities of MntR were determined by using electron paramagnetic resonance (EPR) spectroscopy, as well as competition experiments with the fluorimetric dyes Fura-2 and Mag-fura-2. MntR was not capable of competing with Fura-2 for the binding of transition metal ions. Therefore, the metal-binding affinities and stoichiometries of Mag-fura-2 for Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ were determined and utilized in MntR/Mag-fura-2 competition experiments. The measured Kd values for MntR metal binding are comparable to those reported for DtxR metal binding [Kd from 10(-)7 to 10(-4) M; D'Aquino, J. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 18408-18413], AntR [a homologue from Bacillus anthracis; Sen, K. I. et al. (2006) Biochemistry 45, 4295-4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm-1. This value is lower in magnitude than most known dinuclear Mn2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn2+ site with a longer Mn...Mn distance (4.4 A) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (approximately 160 microM) for its cognate metal ion Mn2+. Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metal-binding affinities of MntR reported here help to elucidate the regulatory mechanism of this metal-dependent transcription factor.
    Preview · Article · Jan 2007 · Biochemistry
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    ABSTRACT: The electronic properties of an unusually redox-rich iron system, [PhBP(R)3]Fe-Nx (where [PhBP(R)3] is [PhB(CH2PR2)3]-), are explored by Mössbauer, EPR, magnetization, and density-functional methods to gain a detailed picture regarding their oxidation states and electronic structures. The complexes of primary interest in this article are the two terminal iron(IV) nitride species, [PhBP(iPr)3]Fe[triple bond]N (3a) and [PhBP(CH2Cy)3]Fe[triple bond]N (3b), and the formally diiron(I) bridged-Fe(mu-N2)Fe species, {[PhBP(iPr)3]Fe}2(mu-N2) (4). Complex 4 is chemically related to 3a via a spontaneous nitride coupling reaction. The diamagnetic iron(IV) nitrides 3a and 3b exhibit unique electronic environments that are reflected in their unusual Mössbauer parameters, including quadrupole-splitting values of 6.01(1) mm/s and isomer shift values of -0.34(1) mm/s. The data for 4 suggest that this complex can be described by a weak ferromagnetic interaction (J/D < 1) between two iron(I) centers. For comparison, four other relevant complexes also are characterized: a diamagnetic iron(IV) trihydride [PhBP(iPr)3]Fe(H)3(PMe3) (5), an S = 3/2 iron(I) phosphine adduct [PhBP(iPr)3]FePMe3 (6), and the S = 2 iron(II) precursors to 3a, [PhBP(iPr)3]Fe-Cl and [PhBP(iPr)3]Fe-2,3:5,6-dibenzo-7-aza bicyclo[2.2.1]hepta-2,5-diene (dbabh). The electronic properties of these respective complexes also have been explored by density-functional methods to help corroborate our spectral assignments and to probe their electronic structures further.
    Preview · Article · Dec 2006 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Cytochrome b6f of oxygenic photosynthesis was studied using multifrequency, multimode EPR Spectroscopy. Frequency dependent signals above g = 4.3, and the observation of parallel-mode signals, are indicative of spin interactions in the complex. We demonstrate the presence of an exchange interaction between the unique high-spin heme cn and a nearby low-spin heme bn, and show that a quinone analog NQNO binds at or near to heme cn. The two hemes remain spin coupled upon the binding of NQNO, though strength of interaction decreases significantly. The electronic coupling implies that the heme bn/cn pair could function as a unit to facilitate 2-electron reduction of plastoquionone without generation of an energetically unfavorable semiquinone intermediate.
    Full-text · Article · Dec 2006 · Journal of the American Chemical Society

Publication Stats

298 Citations
136.83 Total Impact Points

Institutions

  • 2014-2015
    • Northwestern University
      • Department of Chemistry
      Evanston, Illinois, United States
  • 2006-2015
    • Carnegie Mellon University
      • Department of Chemistry
      Pittsburgh, Pennsylvania, United States
  • 2012
    • Emory University
      • Department of Physics
      Atlanta, Georgia, United States