The goal of our research is to determine protein function through the investigation of intermediate state structures generated along a given reaction pathway. These intermediate states involve transient forms of the protein, cofactor and substrate.  In a variety of enzyme systems, intermediates include paramagnetic species in the form of metals and/or organic radicals.  In addition, for systems which lack endogenous paramagnetic species, it is often advantageous to introduce a stable radical "spin label" to serve as a reporter of protein structure and dynamics. Electron paramagnetic resonance (EPR) spectroscopy is well suited for the characterization of all of these classes of paramagnetic species.  Thus our primary experimental tools for structural characterization involve advanced EPR techniques, including electron spin echo envelope modulation (ESEEM), electron nuclear double resonance (ENDOR), and Hyperfine Correlation Spectroscopy (HYSCORE).  Pulsed electron double resonance (PELDOR) techniques are used to measure distances between mutagenically introduced spin labels out to 50 Å.  Quantum mechanical simulations of experimental spectra are developed from first principles for the accurate determination of spectral parameters.  For proteins which are not amenable to NMR or crystallographic techniques, homology modeling and molecular dynamics calculations are used to generate structures which can be tested using EPR techniques.  Instrumentation and spectroscopic techniques are implemented as required by the systems under investigation.  A primary focus in this regard is the development and application of high frequency cw and pulsed EPR/ENDOR spectroscopy.  HF-EPR/ENDOR extends EPR spectroscopy to high magnetic field strengths and enhances the capabilities of the technique to determine molecular and electronic structure.

         Examples of projects currently under study include:  structure of protein active sites and substrate intermediates in coenzyme B₁₂-dependent ribonucleoside triphosphate reductase; identity and structure of radicals generated during catalysis by prostaglandin H synthase (COX or PGHS) and cytochrome c oxidase; molecular structure determination of kinesins using spin-labeling techniques; HF-EPR/ENDOR of a variety of protein and substrate-derived radical species.

      K. I. Sen, H. Wu, J.M. Backer and G.J. Gerfen  “The Structure of p85ni in Class IA Phosphoinositide 3-Kinase Exhibits Interdomain Disorder” Biochemistry  49 2159-66 (2010).

      M.A. Yu, T. Egawa, S.-R. Yeh, D.L. Rousseau and G.J. Gerfen “EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen” J. Magn. Reson.  203 213-19 (2010).

       M.A. Yu, T. Egawa, K. Shinzawa-Itoh, S. Yoshikawa, S-R. Yeh, D.L. Rousseau and G.J. Gerfen  “Radical formation in cytochrome c oxidase”  Bioch. Biophys. Acta 1807 1295-304 (2011) PMID:  21718686.

      J. Manzerova, V. Krymov and G.J. Gerfen  “Investigating the intermediates in the reaction of ribonucleotide triphosphate reductase from Lactobacillus leichmannii:  An application of HF EPR-RFQ technology”  J. Magn. Reson. 213 32-45 (2011).

      M. Yu, T. Egawa, K. Shinzawa-Itoh, S. Yoshikawa, V. Guallar, S.-R. Yeh, D.L. Rousseau and G.J. Gerfen “Two tyrosyl radicals stabilize high oxidation states in cytochrome c oxidase for efficient energy conservation and proton translocation”  J. Am. Chem. Soc. 134 4753-4761 (2012).

       J. Sabat, T. Egawa, C. Lu, D.J. Stehr, G.J. Gerfen, D.L. Rousseau, and S.-R. Yeh “Catalytic intermediates of inducible nitric-oxide synthase stablized by the W188H mutation.”  J. Biol. Chem. 288 6095-6106 (2013).

      N.S. Nemeria, A. Ambrus, H. Patel, G. Gerfen, V. Adam-Vizi, L. Tretter, J. Zhou, J. Wang, and F. Jordan “Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate”  J. Biol. Chem. 289 29859-29873 (2014).

       C.J  Roche., A. Talwar, A.F. Palmer, P. Cabrales, G.J. Gerfen and J.M. Friedman “Evaluating the Capacity to Generate and Perserve Nitric Oxide Bioactivty in Highly Purified Earthworm Erythrocruorin:  A Giant Polymeric Hemoglobin with Potential Blood Substitute Properties.”  J. Biol. Chem. 290 99-117 (2015).

     C. Chatterjee, M.P.M.H. Benoit, V. DePaoli, J.D. Diaz-Valencia, A.B. Asenjo, G.J. Gerfen, D. J. Sharp, H. Sosa "Distinct Interaction Modes of Kinesin-13 Motor Domain in the Microtubule"  Biophys. J. 110 1593-1604  (2016).

     N. S. Nemeria, G.J. Gerfen, E. Guevara, P.R. Nareddy, M. Szostak, F. Jordan  "The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate"  Free Rad. Biol. Med. 108  644-654 (2017).

     A.A. DeColli, N.S. Nemeria, A. Majumdar, G.J. Gerfen, F. Jordan, C. L. Freel Meyers  "Oxidative decarboxylation of pyruvate by 1-deoxy-D-xyulose 5-phosphate synthase, a central metabolic enyzme in bacteria"  J. Biol. Chem. 293 10857-10869 (2018).

     N.S. Nemeria, G.J. Gerfen, L. Yang, and F. Jordan  "Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate deydrogenase on the L-lysine, L-hydroxylysine and L-tryptophan degradation pathways from studies in vitro"    BBA - Bioenergetics  1859 932-939 (2018).