OUR RESEARCH IS FOCUSED primarily on the study of a family of proteins that use derivatives of riboflavin (vitamin B2) as essential cofactors or coenzymes for their biological activity. Our emphasis is the investigation of the structure of the protein, its influence on the chemical and oxidation-reduction and other chemical properties of the bound flavin cofactor, and the role of the cofactor in catalysis and/or electron transfer. Flavoproteins are often an integral part of important electron transport pathways in living systems including such important processes as respiration, photosynthesis, nitrogen fixation, signal transduction, detoxification, DNA repair, and metabolism. Many of these processes represent an important means by which chemical and photochemical energy is transformed and transported within the cell as electro-chemical potential. The flavin cofactor that participates in each of the many redox reactions catalyzed by flavoproteins is essentially the same; however, its properties and chemical reactivity are very different depending on the protein to which it is bound. It is obvious that the protein exerts a major influence on the bound cofactor. How is this accomplished? Our research major goals center around the determination of the protein structural features that are responsible for the modulation of the redox and biochemical properties of the bound flavin cofactor in flavoproteins.

     One of our favorite and most extensively studied model system is the small electron transferase known as the flavodoxin.  Flavodoxins are small proteins containing a non-covalently bound flavin mononucleotide cofactor as their only redox center. These acidic proteins serve as very low potential one-electron transfer proteins in various biological processes including nitrogen-fixation, hydrogen production, sulfate reduction, and photosynthesis in some organisms. This class of flavoprotein is well characterized both biochemically and structurally, providing a good model system in which to investigate how specific molecular interactions between protein and flavin cofactor alter and regulate the oxidation-reduction properties and other physical attributes of the bound cofactor. The effects of hydrogen-bonding, polarity, solvent accessibility, and electrostatic and aromatic interactions are being systematically evaluated by the "re-engineering" of the FMN binding site using recombinant DNA technology.

     More complex flavoproteins, including cytochrome P450 reductase, part of an essential detoxification and xenobiotic-metabolizing system in mammalian cells, and nitric oxide synthetase, an enzyme important in signal transduction contain FMN in addition to the related cofactor (flavin adenine dinucleotide [FAD]). Cytochrome P450 is made up of two principal domains (excluding the membrane-anchoring region)--the FAD-binding domain, which also forms the NADPH binding site, and the FMN-binding domain. Electrons pass from the reduced pyridine nucleotide (NADPH) to the FAD which, in a complex mechanism, are transferred to the FMN. The reductase forms a transient, non-covalent electron transfer complex with cytochrome P450 by which one electron from the FMN passes to the heme-Fe.

     It is of interest and significance that the FMN-binding domains of these more complex flavoproteins are homologous in sequence and tertiary structure to the flavodoxin. It is likely that the flavodoxin serve as a paradigm for the FMN-binding domains in these proteins. However, do they regulate the oxidation-reduction and chemical properties of the cofactor in similar ways? Significant differences are noted between the midpoint potentials of the FMN in cytochrome P450 reductase and the flavodoxin. Thus, another of our long-term goals is to demonstrate whether some of the same basic principles underlying the modulation of the redox properties of the bound cofactor that are found in the simpler bacterial flavodoxin monomer apply directly to these multi-domain systems. Our efforts are primarily focused on studies of the human cytochrome P450 reductase system and the equivalent enzyme from Bacillus megaterium.

     The generically named electron transfer flavoprotein or ETF, provides us with a second system of electron transferase having entirely different properties. These proteins display unusually high oxidation-reduction potentials, among the highest of the flavoprotein family. How does this occur? Composed of two different subunits, these proteins use the flavin adenine dinucleotide (FAD) cofactor. What is the role of each subunit? Besides the FAD cofactor, this protein contains a tightly bound adenosine monophosphate (AMP) molecule. What is its role? Does it affect the redox properties of the flavin? Although our initial efforts have focused on an ETF cloned from a bacterial source, this protein is homologous to related ETF proteins involved in fatty acid oxidation in mammals. Several debilitating diseases have been traced to genetic defects in either the ETF or associated enzymes. Glutaric acidemia type II is one such genetic disorder. This conditions has been traced in part to either ETF deficiencies in these individuals and/or to mutations and polymorphism in the gene encoding for one of the subunits of the ETF protein. Do they involve alterations in the properties of the bound cofactor? Are the redox properties of the FAD cofactor significantly altered? Is the electron-transferring activity of these mutated ETF proteins reduced?

     In all of these projects, we use a variety of protein engineering approaches to alter the flavin-binding site in very specific ways. The mutant proteins are purified and studied, particularly their redox and electron-transferring properties and structure.

     Research techniques used in our research include many recombinant DNA techniques including the cloning, characterization, and manipulation of the structural genes coding for the flavoproteins, de novo gene design and synthesis, site-directed mutagenesis, and heterologous gene expression; protein engineering and protein chemistry technologies including molecular modeling and structural analysis, chemical modification, peptide mapping and purification, peptide sequencing, and amino acid analysis; various spectroscopic methods including ultraviolet/visible, fluorescence, circular dichroism, resonance Raman, and nuclear magnetic resonance spectroscopy; and various biochemical and biophysical methods including redox potentiometry, steady-state kinetics and rapid-reaction (transient) kinetic analyses.

General research interests:

Structure, function, and mechanism of flavoproteins.

Protein structural features responsible for the modulation of the oxidation-reduction potentials and chemical properties and reactivity of flavin cofactors.

Mechanisms of xenobiotic metabolizing and detoxifying flavoenzymes.

Protein structure and dynamics.

Protein engineering, protein design, structural analysis (NMR, X-ray), molecular modeling, and computations.

Protein folding and assembly particularly involving those requiring bound cofactors.

 Last updated, Sept, 2003.