Richard P. Swenson
Research Interests:
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.
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?
Publications
Selected publications from the last 5 years:
Yang KY, Swenson, R.P. (2007) "Modulation of the Redox Properties of the Flavin Cofactor through Hydrogen-Bonding Interactions with the N(5) Atom: Role of alphaSer254 in the Electron-Transfer Flavoprotein from the Methylotrophic Bacterium W3A1." Biochemistry 46(9), 2289-97.
Yang KY, Swenson, R.P. (2007) "Nonresonance Raman Study of the Flavin Cofactor and Its Interactions in the Methylotrophic Bacterium W3A1 Electron-Transfer Flavoprotein." Biochemistry 46(9) , 2298-305.
Murray, T.A., Foster, M.P., and Swenson,
R.P. (2003) "Mechanism
of Flavin Mononucleotide Cofactor Binding to the Desulfovibrio vulgaris Flavodoxin.
2. Evidence for Cooperative Conformational Changes Involving Tryptophan
60 in the Interaction between the Phosphate- and Ring-Binding Subsites." Biochemistry 42,
2317-2327.
Murray, T.A. and Swenson, R.P. (2003) "Mechanism of Flavin Mononucleotide
Cofactor Binding to the Desulfovibrio vulgaris Flavodoxin.
1. Kinetic Evidence for Cooperative Effects Associated with the Binding
of Inorganic Phosphate and the 5'-Phosphate Moiety of the Cofactor." Biochemistry 42,
2307-2316.
Kasim, M. and Swenson, R.P. (2001) "Alanine-Scanning of the 50’s
Loop in the Clostridium beijerinckii Flavodoxin: Evaluation
of Additivity and the Importance of Interactions Provided by the Main
Chain in the Modulation of the Oxidation-Reduction Potentials.", Biochemistry 40,
13548-13555.
Bradley, L. H. and Swenson, R.P. (2001) "Role of Hydrogen Bonding
Interactions to N(3)H of the Flavin Mononucleotide Cofactor in the Modulation
of the Redox Potentials of the Clostridium beijerinckii Flavodoxin.", Biochemistry 40,
8686-8695.
Chang, F.C., Bradley, L. H. and Swenson,
R.P. (2001) "Evaluation
of the Hydrogen Bonding Interactions and Their Effects on the Oxidation-Reduction
Potentials for the Riboflavin Complex of the Desulfovibrio vulgaris Flavodoxin.", Biochimica
et Biophysica Acta 1504, 319-328.
Rotello, V. M. and Swenson, R.P. (2001) “Organic Redox Cofactors.”, Antioxidants
and Redox Signaling 3: 721-722.
Kasim, M. and Swenson, R.P. (2000) "Conformational Energetics
of a Reverse Turn in the Clostridium beijerinckii Flavodoxin
is Directly Coupled to the Modulation of its Oxidation-Reduction Potentials.", Biochemistry 39,
15322-15332.
