Bioinorganic Chemistry of Hemes and Heme Proteins
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 Chemistry and 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).
- 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.