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
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:
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]