Faculty Profile

Faculty Profile of John Enemark

John Enemark

Regents Professor- Emeritus

Email: jenemark@email.arizona.edu
Building: CSML 320
Phone: 520-621-2245

Honors


  • University of Arizona Graduate College and Professional Education Teaching and Mentoring Award, 2002
  • Mortar Board Citation Award, University of Arizona Mortar Board National Senior Honor Society, 2002
  • El Paso Natural Gas Foundation Faculty Achievement Award, 1994
  • Senior Alexander von Humboldt Award (Reinvitation), 1992
  • American Association for the Advancement of Science Fellow, 1991
  • Fulbright Senior Scholar Award, 1989

Education and Appointments


  • B.A. 1962, St. Olaf College
  • A.M. 1964, Harvard University
  • Ph.D. 1966, Harvard University

Research Interests


  • Biochemistry
  • Inorganic
  • Bioinorganic
  • Protein and Membrane Biochemistry
  • Spectroscopy/molecular Structure

Research Summary

Bioinorganic Chemistry; Molybdenum-Containing Enzymes; Electronic Structure and Spectroscopy; Pulsed EPR

Molybdenum is an essential trace element for all forms of life and over 40 molybdoenzymes are known that catalyze oxidation-reduction reactions involved in the metabolism of carbon, nitrogen and sulfur. Of particular interest to our group is the enzyme sulfite oxidase (SO) that is required for normal neurological development in children. The determination of the crystal structure of chicken liver SO from protein prepared in our group has provided a basis for interpreting fatal point mutations in the highly homologous human enzyme. In addition, the novel structure of the molybdenum center of SO provides a target for the synthesis of new active site models as well as a framework for interpreting spectra from the enzyme. The large separation between the molybdenum center and the b-type heme center of SO raises fundamental questions about the process and pathways of intramolecular electron transfer in the protein. Our research involves an integrated program of chemical, biochemical and biophysical studies that include: synthesis of new compounds, X-ray structure determination, theoretical calculations, kinetics, and many types of spectroscopy (CW- and pulsed EPR, electronic, magnetic circular dichroism (MCD), resonance Raman, NMR, K- and L-edge X-ray absorption, photoelectron (PES)).

I. Variable Frequency Pulsed Electron Paramagnetic Resonance (EPR) Spectroscopy

The variable frequency pulsed EPR capabilities at the University of Arizona enable us to directly probe the structures of the transient Mo(V) states that are intermediates in the catalytic cycles of molybdenum enzymes. From Electron Spin Echo Envelope Modulation (ESEEM) and pulsed Electron-Nuclear Double Resonance (ENDOR) methods we have determined the active site structures of SO and DMSO reductase, and are now investigating the effects of point mutations on these structures. Recent advances in pulsed EPR instrumentation in the EPR Facility enable us track oxygen atoms in the catalytic cycle through couplings of Mo(V) to 17-O and 33-S in isotopically enriched frozen solutions. A worldwide network of collaborators provides access to a wide range of molybdenum enzymes and their mutants. The goal of these collaborative studies is to obtain key insight about the catalytic mechanisms of molybdenum enzymes, while simultaneously developing new pulsed EPR methodologies for studying metalloproteins.

II. Kinetics of Intramolecular Electron Transfer

Intramolecular electron transfer (IET) between the molybdenum and heme domains of SO is a key feature of the proposed catalytic mechanism. The wide variation in the rates of intramolecular electron transfer with pH, concentration of anions in the medium, and solution viscosity support a novel mechanism that couples IET to SO conformational changes. IET studies of mutant forms of SO and of related proteins should provide insight into this interesting process and the fundamental factors affecting electron transfer at molybdenum centers in enzymes.

III. Metal-Sulfur Covalency

Metal-sulfur bonds play an important role in many biological and industrial catalysts. We are investigating the covalency of Mo-S bonds by EPR spectroscopy, gas phase UV and X-ray photoelectron spectroscopy, and theoretical calculations. These studies suggest that under appropriate conditions, Mo-S bonds function as an "electronic buffer" to oxidation state changes at the metal center.

IV. Models for Molybdenum-Containing Enzymes

The structure, kinetics and pulsed EPR spectroscopy of SO has stimulated the synthesis of oxo-molybdenum compounds that mimic specific features of its active site. Recently, we have suggested that the "fold-angle" of the ene-1,2-dithiolate ring of the molybdenum cofactor may play an important role in modulating the electronic structure of the molybdenum center and hence in controlling the catalytic process.



Selected Publications

  • Davis, A. C.; Cornelison, M. J.; Meyers, K. T.; Rajapakshe, A.; Berry, R. E.; Tollin, G.; Enemark, J. H. "Effects of Mutating Aromatic Surface Residues of the Heme Domain of Human Sulfite Oxidase on its Heme Midpoint Potential, Intramolecular Electron Transfer, and Steady-State Kinetics." Dalton Trans. in press, (DOI: 10.1039/C2DT31508D). Invited contribution to themed issue on Bioinorganic Chemistry.

  • Rajapakshe, A.; Tollin, G.; Enemark, J. H. "Effects Kinetic and Thermodynamic Effects of Mutations of Human Sulfite Oxidase." Chemistry & Biodiversity, in press, (doi: 10.1002/cbdv.201200010). Invited contribution to Special ICBIC 15 Issue.

  • Klein, E. L.; Astashkin, A. V.; Raitsimring, A. M.; Enemark, J. H. "Applications of Pulsed EPR Spectroscopy to Structural Studies of Sulfite Oxidizing Enzymes." Coord. Chem. Rev., in press,(doi.org/10.1016/j.ccr.2012.05.038), Invited contribution to special issue of in honor of 65th birthday of Prof. E. I. Solomon.

  • Astashkin, A. V.; Rajapakshe, A.; Cornelison, M. J.; Johnson-Winters, K.; Enemark, J. H. "Determination of the Distance Between the Mo(V) and Fe(III) Heme Centers of Wild Type Human Sulfite Oxidase by Pulsed EPR Spectroscopy." J. Phys. Chem. B, 2012, 116, 1942-1950 (DOI: 10.1021/jp210578f).

  • Raitsimring, A.; Astashkin, A.; Enemark, J. H.; Blank, A.; Twig, Y.; Song, Y.; Meade, T. J. . "Di-electric Resonator for Ka-Band Pulsed EPR Measurements at Cryogenic Temperatures. Probehead Construction and Applications." Appl. Magn. Reson., 2012, 42, 441-452, (DOI: 10.1007/s00723-012-0313-1).

  • Klein, E. L; Raitsimring, A. M.; Astashkin, A. V.; Rajapakshe, A.; Johnson-Winters, K.; Arnold, A. R.; Potapov, A.; Goldfarb, D.; Enemark, J. H. "The Identity of the Exchangeable Sulfur-Containing Ligand at the Mo(V) Center of R160Q Human Sulfite Oxidase." Inorg. Chem., 2012, 51, 1408-1418 (DOI: 10.1021/ic201643t).

  • Rajapakshe, A.; Meyers, K. T.; Berry, R. E.; Tollin, G; Enemark, J. H. "Intramolecular Electron Transfer in Sulfite-oxidizing Enzymes: Probing the Role of Aromatic Amino Acids." J. Biol. Inorg. Chem., 2012, 17, 345-352 (DOI: 10.1007/s00775-011-0856-x).

  • Wiebelhaus, N. J.; Cranswick, M. A.; Klein, E. L.; Lockett, L. T.; Lichtenberger, D. L.; Enemark, J. H. "Metal-Sulfur Valence Orbital Interaction Energies in Metal-Dithiolene Complexes: Determination of Charge and Overlap Interaction Energies by Comparison of Core and Valence Ionization Energy Shifts." Inorg. Chem. 2011, 50, 11021-11031 (DOI: 10.1021/ic201566n), November cover article.

  • Rajapakshe, A.; Astashkin, A. V.; Klein, E. L.; Reichmann, D.; Mendel, R. R.; Bittner, F.; Enemark, J. H. "Structural Studies of the Molybdenum Center of Mitochondrial Amidoxime Reducing Component (mARC) by Pulsed EPR Spectroscopy and 17O-Labeling." Biochemistry, 2011, 50, 8813-8822 (DOI: 10.1021/bi2005762).

  • Song, Y.; Meade, T. J.; Astashkin, A. V.; Klein, E. L.; Enemark, J. H.; Raitsimring, A. "Pulsed Dipolar Spectroscopy Distance Measurements in Biomacromolecules Labeled with Gd(III) Markers." J. Magn. Reson. 2011, 210, 59-68 (doi:10.1016/j.jmr.2011.02.010).

  • Enemark, J. H.; Raitsimring, A. M.; Astashkin, A. V.; Klein, E. L. "Implications for the Mechanism of Sulfite Oxidizing Enzymes from Pulsed EPR Spectroscopy and DFT Calculations for "Difficult" Nuclei." Faraday Discuss. 2011, 148, 249-267(DOI: 10.1039/c004404k).

  • Johnson-Winters, K.; Nordstrom, A. R.; Davis, A. C.; Tollin, G.; Enemark, J. H. "Effects of Large Scale Amino Acid Substitution in the Polypeptide Tether Connecting the Heme and Molybdenum Domains on Catalysis in Human Sulfite Oxidase." Metallomics 2010, 2, 766-770(DOI: 10.1039/c0mt00021c).

  • Johnson-Winters, K.; Tollin, G.; Enemark, J. H. "Elucidating the Catalytic Mechanism of Sulfite Oxidizing Enzymes using Structural, Spectroscopic and Kinetic Analyses." Biochemistry 2010, 49, 7242-7254(DOI: 10.1021/bi1008485).

  • Rajapakshe, A.; Johnson-Winters, K.; Nordstrom, A. R.; Meyers, K. T.; Emesh, S.; Astashkin, A. V.; Enemark, J. H. "Characterization of Chloride-Depleted Human Sulfite Oxidase by EPR Spectroscopy: Experimental Evidence for the Role of Anions in Product Release." Biochemistry 2010, 49, 5154-5159 (DOI: 10.1021/bi902172n).

  • Enemark, J. H.; Astashkin, A. V.; Raitsimring, A. M. "High Resolution EPR Spectroscopy of Mo-Enzymes. Sulfite Oxidases: Structural and Functional Implications" in Biological Magnetic Resonance; Vol. 29: Metals in Biology: Applications of High Resolution EPR to Metalloenzymes; Hanson, G. R. and Berliner, L. J., Eds.; Springer, New York, 2010, pp. 121-168.

  • Johnson-Winters, K.; Nordstrom, A. R.; Emesh, S.; Astashkin, A. V.; Rajapakshe, A.; Berry, R. E.; Tollin, G.; Enemark, J. H. "Effects of Interdomain-Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase." Biochemistry 2010, 49, 1290-1296 (DOI: 10.1021/bi9020296).

  • Rapson, T. D.; Astashkin, A. V.; Johnson-Winters, K.; Bernhardt, K. V.; Kappler, U.; Raitsimring, A. M.; Enemark, J. H. "Pulsed EPR Investigations of the Mo(V) Centers of the R55Q and R55M Variants of Sulfite Dehydrogenase from Starkeya novella." J. Biol. Inorg. Chem. 2010, 15, 505-514 (DOI: 10.1007/s00775-009-0619-0).

  • Vannucci, A. K.; Snyder, R. A.; Gruhn, N. E.; Lichtenberger, D. L.; Enemark, J. H. "New Insights into Solvolysis and Reorganization Energy from Gas-Phase, Electrochemical, and Theoretical Studies of Oxo-Tp*Mo(V) Molecules." Inorg. Chem. 2009, 48, 8856-8862 (DOI: 10.1021/ic9011058).

  • Astashkin, A. V.; Klein, E. L.; Ganyushin, D.; Johnson-Winters, K.; Neese, F.; Kappler, U.; Enemark, J. H. "Exchangeable Oxygens in the Vicinity of the Molybdenum Center of the High-pH Form of Sulfite Oxidase and Sulfite Dehydrogenase." Phys. Chem. Chem. Phys. 2009, 11,6733-6742 (DOI:10.1039/b907029j).

  • Rajapakshe, A.; Snyder, R. A.; Astashkin, A. V.; Bernardson, P.; Evans, D. J.; Young, C. G.; Evans, D. H.; Enemark, J. H. "Insight into the Nature of Mo(V) Species in Solution: Modeling Catalytic Cycles for Molybdenum Enzymes." Inorg. Chim. Acta 2009, 362, 4603-4608 (doi: 10.1016/j.ica.2009.05.040).

  • Klein, E. L.; Astashkin, A. V.; Ganyushin, D.; Riplinger, C.; Johnson-Winters, K.; Neese, F.; Enemark, J. H. "Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using ESEEM Spectroscopy, Isotopic Labeling, and DFT Calculations." Inorg. Chem. 2009, 48, 4743-4752 (DOI:10.1021/ic801787s).

  • Emesh, S.; Rapson, T. D.; Rajapakshe, A.; Kappler, U.; Bernhardt, P. V.; Tollin, G.; Enemark, J. H. "Intramolecular Electron Transfer in Sulfite Oxidizing Enzymes: Elucidating the Role of a Conserved Active Site Arginine." Biochemistry 2009, 48, 2156-2163 (DOI: 10.1021/bi801553q).

  • Bailey, S.; Rapson, T.; Johnson-Winters, K.; Astashkin, A. V.; Enemark, J. H.; Kappler, U. "Molecular Basis for Enzymatic Sulfite Oxidation: How Three Conserved Active Site Residues Shape Enzyme Activity." J. Biol. Chem. 2009, 284, 2053-2063 (doi:10.1074/jbc.M807718200).

  • Cranswick, M. A.; Gruhn, N. E.; Enemark, J. H.; Lichtenberger, D. L. "Electronic Structure of the d¹ Bent metallocene Cp2VCl2: A Photoelectron and Density Functional Study." J. Organometal. Chem. 2008, 693, 1621-1627 (doi.org/10.1016/j.jorganchem.2007.12.035).

  • Astashkin, A. V.; Johnson-Winters, K.; Klein, E. L.; Feng, C.; Wilson, H. L.; Rajagopalan, K. V.; Raitsimring, A. M.; Enemark, J. H. "Structural Studies of the Molybdenum Center of the Pathogenic R160Q Mutant of Human Sulfite Oxidase by Pulsed EPR Spectroscopy and 17O and 33S Labeling." J. Am. Chem. Soc. 2008, 130, 8471-8480 (DOI: 10.1021/ja801406f).

  • Enemark, J. H.; Astashkin, A. V.; Raitsimring, A. M. "Structures and Reaction Pathways of the Molybdenum Centers of Sulfite Oxidizing Enzymes by Pulsed EPR Spectroscopy." Biochem. Soc. Trans. 2008, 36, 1129-1133 (doi:10.1042/BST0361129).

  • Astashkin, A. V.; Johnson-Winters, K.; Klein, E. L.; Byrne, R. S.; Hille, R.; Raitsimring, A. M.; Enemark, J. H. "Direct Demonstration of the Presence of Coordinated Sulfate in the Reaction Pathway of Arabidopsis thaliana Sulfite Oxidase Using 33S Labeling and ESEEM Spectroscopy." J. Am. Chem. Soc. 2007, 129, 14800-14810.

  • Feng, C.; Tollin, G.; Enemark, J. H. "Sulfite Oxidizing Enzymes." Biochim. Biophys. Acta 2007, 1774, 527-539.

  • Enemark, J. H.; Astashkin, A. V.; Raitsimring, A. M. "Investigation of the Coordination Structures of the Molybdenum(V) Sites of Sulfite Oxidizing Enzymes by Pulsed EPR Spectroscopy." Dalton Trans. 2006, 3501-3514.

  • Inscore, F. E.; Knottenbelt, S.; Rubie, N. D.; Joshi, H. K.; Kirk, M. L.; Enemark, J. H. "Understanding the Origin of Metal-Sulfur Vibrations in an Oxo-Molybdenum Dithiolene Complex: Relevance to Sulfite Oxidase." Inorg. Chem. 2006, 45, 967-976.

  • Astashkin, A. V.; Feng, C.; Raitsimring, A. M.; Enemark, J. H. "17O ESEEM Evidence for Exchange of the Axial Oxo Ligand in the Molybdenum Center of the High pH Form of Sulfite Oxidase." J. Am. Chem. Soc. 2005, 127, 502-503.

  • Astashkin, A. V.; Neese, F.; Raitsimring, A. M.; Cooney, J. J. A.; Bultman, E.; Enemark, J. H. "Pulsed EPR Investigations of Systems Modeling Molybdenum Enzymes: Hyperfine and Quadrupole Parameters of Oxo-17O in [Mo17O(SPh)4]-." J. Am. Chem. Soc. 2005, 127, 16713-16722.

  • Astashkin, A. V.; Hood, B. L.; Feng, C.; Hille, R.; Mendel, R. R.; Raitsimring, A. M.; Enemark, J. H. "Structures of the Mo(V) Forms of Sulfite Oxidase from Arabidopsis thaliana by Pulsed EPR Spectroscopy." Biochemistry, 2005, 44, 13274-13281.

  • Feng, C.; Wilson, H. L.; Tollin, G.; Astashkin, A. V.; Hazzard, J. T.; Rajagopalan, K. V.; Enemark, J. H. "The Pathogenic Human Sulfite Oxidase Mutants G473D and A208D are Defective in Intramolecular Electron Transfer." Biochemistry 2005, 44, 13734-13743.

  • Enemark, J. H.; Cooney, J. J. A.; Wang, J-J.; Holm, R. H. "Synthetic Analogues and Reaction Systems Relevant to the Molybdenum and Tungsten Oxotransferases." Chem. Rev. 2004, 104, 1175-1200.


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