Areas of Interest
1. The research interests of Dr. Schenkman are mainly in
the area of the cytochrome P450-containing monooxygenase system.
The role of cytochrome b5 as an enhancer of the
monooxygenase reaction and its site of interaction on P450 is
under investigation. Studies in this laboratory involve
determination of the nature of interaction between the proteins
of the monooxygenase, including cytochrome P450,
NADPH-cytochrome P450 reductase, and cytochrome b5.
Perturbants of the interaction, including alteration of
conductivity of the medium using different ionic strength and
inclusion of polyols, as well as pH variation have been used to
determine the nature of forces driving the interaction. The
influence of these perturbants on electron transfer between the
proteins and the direction of electron transfer are examined.
Stopped flow spectroscopy of electron transfer reactions are
utilized as well as product identification in the processes.
Based upon studies in this laboratory the hypothesis was
raised that cytochrome b5 serves as an electron
buffer, taking an electron from reduced cytochrome P450, then
returning it after a second electron reduces the cytochrome P450
and oxygen binds to it (See sketch at left). The prosthetic
group of hemoproteins is a relatively small 616 Da, compared
with the mass of the native cytochrome b5 (17 kDa) or
cytochrome P450 (50 kDa) (see below). Consequently, a mechanism
for precise alignment of the redox centers of the two proteins
would be needed for increased efficiency of electron transfer.
The interaction between cytochrome P450 and cytochrome b5
has been shown to be by complementary charge-pairing involving
conserved acidic residues of cytochrome b 5. This interaction
generally stimulates the monooxygenase turnover of substrates.
The factors responsible for the enhanced turnover are of special
interest. Dr. Schenkman is interested in the topology of
interaction between NADPH-cytochrome P450 reductase and
cytochrome P450 and between cytochrome b5 and
cytochrome P450. A goal is to try to try to track the route of
electron transfer from the surface of the cytochrome P450 to its
buried heme prosthetic group. Dr. Schenkman has recently
published a review on cytochrome b5 :J.B. Schenkman
and I Jansson, The many roles of cytochrome b5,
Pharmacology & Therapeutics 97:139-152 (2003),
http://p450.uchc.edu/papers.html.
2. Cytochrome b5 is an acidic protein. 25
of its 131 amino acid residues is either an aspartic acid or
glutamic acid residue. A number of these acidic residues are
located around a cleft in the protein in which the heme
prosthetic group resides. Figure 1 shows the crystal structure
of this protein. Several of the acidic residues around the heme
edge, plus one of the heme propionate residues, has been shown
to participate in complementary charge-pairing with redox
partners of cytochrome b5. The structure of
cytochrome P450cam (CYP101) is known, and the heme is located
under a shallow depression on the proximal surface of the
protein. It is believed that all of the forms of cytochrome P450
might have a similar structure. Above the heme of CYP101 are a
number of cationic amino acids which could serve as potential
charge-pairing residues for an acidic reduction/oxidation
partner like cytochrome b5.
Modeling of putative interactions between potential
charge-pairing residues suggested the site of cyt b5
interaction on CYP101 is on the proximal surface over the heme.
Competition of cyt b5 with putidaredoxin, the native
electron transfer protein to CYP101, and site directed
mutagenesis of putative charge-pairing residues of both proteins
supported the model. Sequence alignments and structural
alignments of different mammalian forms of cytochrome P450 with
CYP101 have been made, and putative residues that might
charge-pair with cytochrome b5 acidic residues of
mammalian are being determined. Figure 2a and b show the surface
of CYP101 proximal to the heme. We have shown that cytochrome b5
binding is at a site over the heme of P450 (light blue or cyan
color on right), charge-pairing with cationic (dark blue) amino
acid residues. Our studies have made use of quartz crystal
microbalance measurements of cytochrome P450 binding to solid
supports (Figure 3) and the binding of cytochrome b5
to the P450. We have also used atomic force microscopy to view
molecules of cytochrome P450 binding to lipid surfaces. These
findings were reported as, Schenkman, et al., 'Charge-dependent
sidedness of cytochrome P450 forms studied by quartz crystal
microbalance and atomic force microscopy.' Archives of
Biochemistry and Biophysics 385:78-87, 2001.
3. In other studies, Dr. Schenkman and his group have
noted that a number of forms of cytochrome P450 are found in the
developing embryo and fetus (Choudhary, et al., Comparative
expression profiling of 40 mouse cytochrome P450 genes in
embryonic and adult tissues." Archives of Biochemistry and
Biophysics 414:91-100 2003), and have hypothesized that these
forms are involved in normal development ("Involvement of
cytochrome P450 in development", Proc. Indian Natn. Sci Acad.
B69 (6): 939-941, 2003)
http://p450.uchc.edu/papers.html. We
have developed heterologous expression systems using Escherichia
coli for production of different cytochrome P450 forms in order
to determine their properties and their possible involvement in
a number of genetic disorders in humans. The absence of a
functional form of cytochrome 450 1B1 (CYP1B1) has beenlinked to
a disease phenotyope, Primary Congenital Glaucoma (PCG) in
humans. We have prepared this hemoprotein and studies its
characteristics and believe there is a role for this enzyme in
normal eye development. The PCG phenotype is characterized as
improper development of the trabecular meshwork, which serves as
a filter for fluids of the anterior chamber of the eye. The
improper development results in poor drainage of this chamber
and pressure builds up that if not aleviated damages the retina
and results in blindness. Mice have an orthologous form of this
hemoprotein, Cyp1b1, and the Cyp1b1 -/- (knockout) mouse has
similar defects in the trabecular meshwork. We examined two
mutations of CYP1B1, where incomplete penetrance in the PCG
phenotype is seen. Each of these forms of cytochrome P450 had a
single point mutation that altered only one amino acid; G61E and
R469W. In the former a neutral amino acid, glycine, at position
61 is converted to an acidic amino acid, glutamic acid. In the
other mutation a basic amino acid, arginine is converted to a
hydrophobic amino acid, tryptophan. We were able to show that
the resultant forms of cytochrome P450 were expressed, but had
considerably lower activity than the wild type enzyme. It was
suggested that a possible reason for the incomplete penetrance
with these forms was the influence of environmental factors that
influenced the expression levels of the proteins, elevations
which enabled the individual to have a normal eye development.
Jansson, et al., "Effect of two mutations of humamn CYP1B1, G61E
and R469W, on stability and endogenous steroid substrate
metabolism." Pharmacogenetics 11:1-9, 2001.
http://p450.uchc.edu/papers.html
4. Currently, we are studying several forms of
cytochrome P450 that have orthologs in other mammalian and
vertebrate species for a possible role in psoriasis. At least
two forms are known to be expressed in epithelial cells, and one
form, CYP2S1 has been found in psoriatic skin to be elevated to
levels considerably higher than adjacent, uninvolved skin. We
believe it is possible that altered expression of certain forms
of cytochrome P450 could cause enhanced growth and development
or regions of the skin, as well as influence the inflammatory
processes seen with this disease.
Publications
Selected Publications
Sarfarazi M, Stoilov I, Schenkman JB. Genetics and biochemistry
of primary congenital glaucoma. Ophthalmol Clin North Am. 2003
Dec;16(4):543-54, vi. Review.
Choudhary D, Jansson I, Schenkman JB, Sarfarazi M, Stoilov I.
Comparative expression profiling of 40 mouse cytochrome P450
genes in embryonic and adult tissues. Arch Biochem Biophys. 2003
Jun 1;414(1):91-100.
Estavillo C, Lu Z, Jansson I, Schenkman JB, Rusling JF.
Epoxidation of styrene by human cyt P450 1A2 by thin film
electrolysis and peroxide activation compared to solution
reactions. Biophys Chem. 2003 May 1;104(1):291-6.
Zhou L, Yang J, Estavillo C, Stuart JD, Schenkman JB, Rusling
JF. Toxicity screening by electrochemical detection of DNA
damage by metabolites generated in situ in ultrathin DNA-enzyme
films. J Am Chem Soc. 2003 Feb 5;125(5):1431-6.
Schenkman JB, Jansson I. The many roles of cytochrome b5.
Pharmacol Ther. 2003 Feb;97(2):139-52. Review.
Munge B, Estavillo C, Schenkman JB, Rusling JF. Optimization
of electrochemical and peroxide-driven oxidation of styrene with
ultrathin polyion films containing cytochrome P450cam and
myoglobin. Chembiochem. 2003 Jan 3;4(1):82-9.
Jansson I, Stoilov I, Sarfarazi M, Schenkman JB. Effect of
two mutations of human CYP1B1, G61E and R469W, on stability and
endogenous steroid substrate metabolism. Pharmacogenetics. 2001
Dec;11(9):793-801.
Schenkman JB, Jansson I, Lvov Y, Rusling JF, Boussaad S, Tao
NJ. Charge-dependent sidedness of cytochrome P450 forms studied
by quartz crystal microbalance and atomic force microscopy. Arch
Biochem Biophys. 2001 Jan 1;385(1):78-87.
Stoilov I, Jansson I, Sarfarazi M, Schenkman JB. Roles of
cytochrome p450 in development. Drug Metabol Drug Interact.
2001;18(1):33-55. Review.
Jansson I, Stoilov I, Sarfarazi M, Schenkman JB. Enhanced
expression of CYP1B1 in Escherichia coli. Toxicology. 2000 Apr
3;144(1-3):211-9.
Lu Z, Lvov Y, Jansson I I, Schenkman JB, Rusling JF.
Electroactive Films of Alternately Layered Polycations and
Iron-Sulfur Protein Putidaredoxin on Gold. J Colloid Interface
Sci. 2000 Apr 1;224(1):162-168.
Rusling JF, Zhou L, Munge B, Yang J, Estavillo C, Schenkman
JB. Applications of polyion films containing biomolecules to
sensing toxicity. Faraday Discuss. 2000;(116):77-87; discussion
171-90.
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