John B. Schenkman
Professor
Department of Cell Biology
University of Connecticut Health Center
Farmington, CT 06032
phone 860 679 3694
fax 860 679 2473
email jschenkm@neuron.uchc.edu
Education
B.A., Brooklyn College
Ph.D., State University of New York
Research interests
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 servcs 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 relatively 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 b5. This interaction generally stimulates the monooxygenase turnover of substrates. The factors responsible for the enhanced turnover are of especial interest. The laboratory 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.
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.
Fig. 1. Crystal structure of cytochrome b5 showing acidic residues around the heme cleft.
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. In this laboratory CYP2B4, the rabbit liver microsomal hemoproteins, are produced in an E.coli heterologous expression system which is also used to prepare mutants of the protein for tracking of the electron flow routes. In other studies, work is proceeding on implementing expression of this protein as well as human forms of cytochrome P450 for studies on Primary Congenital Glaucoma.
Figure 3. Atomic force microscopy of binding of cytochrome P450 2B4 and cytochrome b5 to polyion polymer surfaces on a solid (gold) substrate. The darker the color the closer the molecules are to the bottom (gold) surface. Figure 3. Atomic force microscopy of binding of cytochrome P450 2B4 and cytochrome b5 to polyion polymer surfaces on a solid (gold) substrate. The darker the color the closer the molecules are to the bottom (gold) surface.
Department of Cell Biology Faculty
