Faculty

Research: Experimental biophysics.

Our research programs are focused on structural and functional studies of iron-containing proteins, which catalyze a variety of important biological reactions. Physical techniques that are sensitive to the iron electronic structure, such as Mössbauer and EPR spectroscopies, are employed. Currently, our efforts are concentrated on the studies of two important classes of enzymes: carboxylate-bridged diiron enzymes and iron-sulfur proteins.

Proteins that contain carboxylate-bridged diiron centers form a class of enzymes that activate O₂ for diverse biological functions. These include the oxygen carrier hemerythrin, the R2 subunit of the aerobic Fe-containing ribonucleotide reductase, the hydroxylase component of the soluble methane monooxygenase, the stearoyl-acyl carrier protein delta-9 desaturase, alkane -hydroxylase, xylene monooxygenase, phenol hydroxylase, and the iron storage protein ferritin. We have applied rapid freeze-quench techniques together with Mössbauer spectroscopy to investigate the oxygen­activation mechanisms involved in (1) the generation of the catalytically important tyrosyl radical (Y122·) in the R2 subunit of ribonucleotide reductase from Escherichia coli, (2) the reaction of O₂ with the diferrous form of the methane monooxygenase hydroxylase from Methylococus capsulatus (Bath), and (3) the ferroxidase reaction of recombinant frog ferritins. Several novel, kinetically competent, transient intermediates have been identified and characterized, both spectroscopically and kinetically, providing direct mechanistic insights into these important biological processes.

Iron-sulfur proteins are a group of functionally diverse proteins that contain prosthetic groups composed of Fe and inorganic sulfur of various structures, termed Fe-S clusters. In addition to the well-established role of electron transport, Fe-S proteins are involved in a diverse range of non-redox processes including sensing and regulatory functions. In our laboratory, we employ a combined spectroscopic/rapid-kinetic approach to investigate two of the most interesting recent developments in Fe-S protein research, namely, the biosynthesis of Fe-S clusters and the newly discovered functional role of Fe-S cluster in stabilizing radical intermediates. Detailed mechanistic insights at a molecular level are expected to emerge from our investigations.

Selected Recent Publications:

1. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase.
   J. Baldwin, W. C. Voegtli, N. Khidekel, P. Moënne-Loccoz, C. Krebs, A. S. Pereira, B. A. Ley, B. H. Huynh, T. M. Loehr, P. J. Riggs-Gelasco, A. C. Rosenzweig, J. M. Bollinger, Jr. (2001) J. Am. Chem. Soc., 123, 7017-7030.[abstract] [full text]

2. IscA, an Alternate Scaffold for the Fe-S Cluster Biosynthesis.
   C. Krebs, J. N. Agar, A. D. Smith, J. Frazzon, D. R. Dean, B. H. Huynh, and M. K. Johnson (2001) Biochemistry, 40, 14069-14080. [full text]

3. Coordination of Adenosylmethionine to a Unique Iron Site of the [4Fe-4S] of Pyruvate Formate Lyase Activating Enzyme: A Mössbauer Spectroscopic Study.
   C. Krebs, W. E. Broderick, T. F. Henshaw, J. B. Broderick, and B. H. Huynh (2002) J. Am. Chem. Soc., 124, 912-913.[abstract] [full text]

4. Functional Mimic of Dioxygen-Activating Centers in Non-Heme Diiron Enzymes: Mechanistic Implications of Paramagnetic Intermediates in the Reactions between Diiron(II) Complexes and Dioxygen.
   D. Lee, B. Pierce, C. Krebs, M. P. Hendrich, B. H. Huynh, and S. J. Lippard, (2002) J. Am. Chem. Soc., 124, 3993-4007. [abstract] [full text]

5. Exchange Coupling Constant J of Peroxodiferric Reaction Intermediates determined by Mössbauer Spectroscopy..
   C. Krebs, J. M. Bollinger, Jr., E. C. Theil, and B. H. Huynh (2002) J. Biol. Inorg. Chem., 7, 863-869. [full text]

6. Rapid Electron Transfer during Formation of Cluster X and Kinetic Competence of the Intermediate for Tyrosyl Radical Production in Protein R2 of Ribonucleotide Reductase from Mouse.
   D. Yun, C. Krebs, G. P. Gupta, D. F. Iwig, B. H. Huynh, and J. M. Bollinger, Jr., (2002) Biochemistry, 41, 981-990. [full text]