Bioinorganic Chemistry of Hemes and Heme Proteins; Porphyrin Synthesis, Site-Directed Mutagenesis, Electrochemistry, NMR and EPR Spectroscopies.
We study a wide range of bioinorganic and biological
systems, all of which fall under the general theme of gaining a better understanding of the heme centers in heme proteins that are vital to the life of almost all living organisms. The overall goals of this research are:
To characterize
the nitrosylheme proteins from blood-sucking insects. Nitric
oxide has been shown to be an important neurotransmitter,
vasodilator, and other chemical messenger. We have recently shown
that Rhodnius prolixus (the “kissing bug”)1,2
and Cimex lectularius (the bedbug)3 each have at
least one NO-carrying heme protein in their saliva that helps them
succeed in their goal of living on the blood of higher animals. In
collaboration with the research group of Dr. William Montfort, Dept.
of Biochemistry, and others off-campus, we are investigating the 3-D
structures, spectroscopy (NMR, EPR, UV-vis, MCD, Mössbauer and
resonance Raman), thermodynamics (kinetics and equilibria of NO
binding, and reduction potentials in the absence and presence of NO)
of the NO-binding heme proteins from both of these insects4. Current research on these proteins includes preparation and investigation of appropriate site-directed mutants to test hypotheses as to which amino acid side chains affect NO and histamine binding and release.
To prepare and investigate site-directed mutants of the membrane-bound cytochrome b of the bc1 complex (respiratory Complex III) of the photosynthetic bacterium Rhodobacter sphaeroides. The mutants are chosen so as to interfere with the binding of one of the histidine ligands of either the high- or the low-potential heme of cytochrome b, in order to determine how this interference affects the EPR spectrum and the reduction potential of that heme, and the rate of electron transfer through the low-potential half of the bifurcated electron transfer pathway of the Q-cycle.
To investigate
the reaction mechanisms, substrate specificity and domain
interactions of cytochrome P450BM3 and several insect cytochromes
P450. Cytochromes P450 are monooxygenase enzymes that comprise
about 10% of the protein found in the human liver. They hydroxylate
or epoxidize a wide range of substrates including (but not limited
to) steroid hormones and xenobiotics. P450BM3, from Bacillus
megaterium, has been described as a “complete enzyme”
because in one polypeptide it has both the heme and reductase
domains necessary to catalyze the oxidation of substrates in the
presence of molecular oxygen and NADPH, and its reactions are
100-1000 times faster than those of reconstituted microsomal P450s.
We are using the “scanning chimeragenesis” approach to create
P450BM3 mutants with altered substrate specificity. Techniques
being used include genetic engineering, protein expression and
purification, optical, multidimensional NMR, EPR and pulsed EPR
spectroscopies, stopped-flow kinetics, gas chromatography and mass
spectrometry (for identification of the products of enzymatic
reactions).
To investigate
model hemes and heme proteins by multidimensional NMR spectroscopy.
The goal of these studies is to use (and to further develop) modern
NMR techniques, such as COSY, NOESY, TOCSY, ROESY, HOESY, HMQC,
HMBC, etc., to determine the solution structure and investigate the
dynamic reactions (including axial ligand rotation, macrocycle
inversion and ligand exchange) of selected model heme complexes in
which the oxidation state of iron ranges from +1 to +4, all of which
have relevance to the biological roles of hemes and heme proteins.
Computer modeling of the structural and dynamic features of these
molecules will continue to complement the studies.
- J.M.C. Ribeiro, J.M.H. Hazzard, R.
Nussenzveig, D. Champagne, F.A. Walker, Science 1993, 260, 539.
- J.M.C. Ribeiro, F.A. Walker, J. Exper. Med. 1994, 180, 2251.
- J.G. Valenzuela, F.A. Walker, J.M.C. Riberiro, J. Exper. Biol. 1995, 198, 1519.
- F.A. Walker, J. Inorg Biochem. 2005, 99, 216-236.
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- “Chimeragenesis
of the Fatty Acid Binding Site of Cytochrome P450BM3. Replacement of
the Residues 73-82 with the Homologous Residues from the Insect
Cytochrome P450 CYP4C7”, Murataliev, M. B.; Trinh, L. N.; Moser, L.
V.; Bates, R. B.; Feyereisen, R.; Walker, F. A. Biochemistry
2004, 43, 1771-1780.
- “Models of the Bis-Histidine-Ligated Electron-Transferring Cytochromes. Comparative Geometric and Electronic Structure of Low-Spin Ferro- and Ferrihemes”, Walker, F. A. Chem. Rev.
2004, 104, 589-615.
- “Low-Spin Ferriheme Models of the Cytochromes: Mössbauer Spectra of Six Crystalline Complexes Having Axial Ligand Plane Dihedral Angles Ranging from 0o to 90o”, Teschner, T.; Yatsunyk, L. A.; Schünemann, V.; Paulsen, H.; Winkler, H.; Hu, C.; Scheidt, W. R.; Walker, F. A.; Trautwein, A. X. J. Am. Chem. Soc. 2006, 128, 1379-1389.
- “Models of the Cytochromes: Crystal Structures and EPR Spectral Characterization of Low-Spin Bis-Imidazole Complexes of (OETPP)FeIII Having Intermediate Ligand Plane Dihedral Angles”, Yatsunyk, L. A.; Dawson, A.; Carducci, M. D.; Nichol, G. S.; Walker, F. A. Inorg. Chem.
2006, 45, 5417-5428.
- “Assignment of the Ferriheme Resonances for the High-Spin Form of Nitrophorins 1 and 4 by 1H and 13C NMR Spectroscopy: Comparison to Structural Data Obtained from X-Ray Crystallography”, Shokhireva, T. Kh.; Smith, K. M.; Berry, R. E.; Shokhirev, N. V.; Balfour, C.; Zhang, H.; Walker, F. A. Inorg. Chem.
2007, 46, 2041-2056.
- “Overexpression in Escherichia coli and Functional Reconstitution of the Liposome Binding Ferriheme Protein Nitrophorin 7 (NP7) from the Blood Sucking Bug Rhodnius prolixus”, Knipp, M.; Zhang, H.; Berry, R. E.; Walker, F. A. Prot. Expr. Purif.
2007, 54, 183-191.
- “Effect of the N-Terminus on Heme Cavity Structure, Ligand Equilibrium and Rate Constants, and Reduction Potentials of Nitrophorin 2 from Rhodnius prolixus”, Berry, R. E.; Shokhireva, T. Kh.; Filippov, I.; Shokhirev, M. N.; Zhang, H.; Walker, F. A. Biochemistry
2007, 46, 6830-6843.
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