Faculty Directory

John Enemark

Regents Professor Emeritus - Retired
Degrees and Appointments: 
B.A. 1962, St. Olaf College
A.M. 1964, Harvard University
Ph.D. 1966, Harvard University
University of Arizona,, Assistant Professor of Chemistry, 1968-72
Associate Professor,1972-77, Professor, 1977-98
Regents Professor, 1998-2006
Regents Professor Emeritus, 2006 - present
Awards and 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
Fulbright Senior Scholar Award, 1989
American Association for the Advancement of Science Fellow, 1971
Research Summary: 

1. Molybdenum-containing Enzymes

Molybdenum is an essential trace element for all forms of life and over 50 molybdoenzymes are known that catalyze oxidation-reduction reactions involved in the metabolism of carbon, nitrogen and sulfur. The enzyme sulfite oxidase (SO) is required for normal neurological development in children, and 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 has raised fundamental questions about the process and pathways of intramolecular electron transfer in the protein. Our research has involved 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, and photoelectron (PES)) (see publications).


Structure of Chicken Sulfite Oxidase

 

Sulfite Oxidase Active Site

 

Sulfite Oxidase Proposed Mechanism

 

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

The variable frequency pulsed EPR capabilities at the University of Arizona have enabled 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 the effects of point mutations on these structures. Advances in pulsed EPR instrumentation in the EPR Facility enabled us to 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 has provided access to a wide range of molybdenum enzymes and their mutants. The goal of these collaborative studies has been to obtain key insight about the catalytic mechanisms of molybdenum enzymes, while simultaneously developing new pulsed EPR methodologies for studying metalloproteins.

3. Measurement of Long Distances in Macrobiomolecules 

Double electron-electron resonance (DEER) spectroscopy and Gd(III) spin-labels have been developed in collaboration with Arnold Raitsimring of the EPR Facility, Professor D. Goldfarb of the Weizmann Institute and Professor T. Meade of Northwestern University, to measure long distances (up to 100 Ångstroms) in macrobiomolecules.  Shown below is an initial example of distance determination of ~60 Å in a 14-mer of DNA (Journal of Magnetic Resonance 210 [2011] 59–68).

DEER of GD(III)-labeled DNA

Publications: 

Selected publications, for a complete list see the Enemark Group page.

Kappler, U.; Enemark, J. H. “Sulfite Oxidizing Enzymes.” J. Biol. Inorg. Chem. 2015, 20, 253-264 (DOI 10.1007/s00775-014-1197-3).

Davis, A. C.; Johnson-Winters, K.; Arnold, A. R.; Tollin, G.; Enemark, J. H. "Kinetic Results for Mutations of Conserved Residues H304 and R309 of Human Sulfite Oxidase Point to Mechanistic Complexities." Metallomics, 2014, 6, 1664-1670 (DOI: 10.1039/C4MT00099D).

Klein, E. L.; Belaidi, A. A.; Raitsimring, A. M.; Davis, A. C.; Krämer, T.; Astashkin, A. V.; Neese, F.; Schwarz, G.; Enemark, J. H. "Pulsed Electron Paramagnetic Resonance Spectroscopy of 33S Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase." Inorg. Chem., 2014, 53, 961−971 (DOI: 10.1021/ic4023954).

Johnson-Winters, K.; Davis, A. C.; Arnold, A. R.; Berry, R. E.; Tollin, G.; Enemark, J. H. "Probing the Role of a Conserved Salt Bridge in the Intramolecular Electron Transfer Kinetics of Human Sulfite Oxidase." J. Biol. Inorg. Chem., 2013, 18, 645-653 (DOI: 10.1007/s00775-013-1010-8).

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., 2013, 42, 3043-3049 (DOI: 10.1039/C2DT31508D). Invited contribution to themed issue on Bioinorganic Chemistry.

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., 2013, 257, 110-118 (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.

Rajapakshe, A.; Tollin, G.; Enemark, J. H. "Kinetic and Thermodynamic Effects of Mutations of Human Sulfite Oxidase." Chemistry & Biodiversity, 2012, 9, 1621-1634 (doi: 10.1002/cbdv.201200010). Invited contribution to Special ICBIC 15 Issue.

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).