J. Robert Reed, PhD

Research

The cytochromes P450 (P450) are heme-containing enzymes expressed in virtually all tissues and species and are responsible for the primary metabolism of most drugs and other xenobiotics. The enzymes are members of a superfamily, and most of the mammalian isoforms display a broad range of substrate specificity. Catalysis by these enzymes relies on a complicated reaction mechanism involving the delivery of electrons from a di-flavin-containing redox partner (the NADPH cytochrome P450 reductase) and/or another heme protein, the cytochrome b5.

The binding between P450 reductase and P450 is extremely complicated. Hydrophobic and electrostatic interactions have been proposed as the driving force for the interaction between the enzymes. In addition, other electrostatic interactions also have been shown to be competitive to the binding process. Despite over 20 years of research, there remains no general consensus regarding the nature of the interaction between the proteins. From the findings of many studies, a number of charged amino acid residues have been implicated in the binding between the two proteins. Furthermore, there have been conflicting studies regarding the binding site on the surface of P450 for P450 reductase and cytochrome b5. Whereas, some studies have indicated that cytochrome b5 and P450 reductase can bind at the same time to P450, others have shown that the binding sites overlap for the two proteins.

The findings of recent publications have suggested that the interactions and binding between P450 and its redox partners, especially the P450 reductase, may be a dynamic process which represents specific changes in the orientation and perhaps the loci of contact at the different steps of the catalytic cycle. Furthermore, at least two modes of P450 reductase and P450 interactions have been associated with different substrates. My work involves using site-directed mutagenesis, covalent cross-linking agents, and stopped flow spectrophotmetry (a technique that can discern pre-steady state spectral changes resulting from enzyme-enzyme interactions) to understand the nature of the interaction between P450 reductase and P450 at the different stages of the P450 catalytic cycle and with different substrates of P450.

References

Reed, J. R. and Hollenberg, P. F. (2003) New Perspectives on the Conformational Equilibrium Regulating Multi-Phasic Reduction of Cytochrome P450 2B4 by Cytochrome P450 Reductase. J. Inorg. Biochem. 97: 276-286.

Reed, J. R. and Hollenberg, P. F. (2003) Examining the Mechanism of Stimulation of Cytochrome P450 by Cytochrome b5: The Effect of Cytochrome b5 on the Interaction Between Cytochrome P450 and P450 Reductase. J. Inorg. Biochem. 97: 265-275.

Reed, J. R. and Hollenberg, P. F. (2003) Comparison of Substrate Metabolism by Cytochromes P450 2B1, 2B4, and 2B6: Relationship of Heme Spin State, Catalysis, and the Effects of Cytochrome b5. J. Inorg. Biochem. 93: 152-160.

Hanna, I. H.; Reed, J. R.; Guengerich, F. P.; and Hollenberg, P. F. (2000) Expression of Human Cytochrome P450 2B6 in Escherichia coli: Characterization of Catalytic Activity and Expression Levels in Human Liver. Arch. Biochem. Biophys. 376: 206-216.