James R. Reed
Research Assistant Professor
Ph.D., University of Nevada, Reno, 1995
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 cancer-causing xenobiotics. The enzymes are members of a gene superfamily. Most of the mammalian isoforms are bound to the endoplasmic reticulum and display a broad range of substrate specificity. In order to serve its role in first pass metabolism, the liver expresses a variety of different types of P450 enzymes. Catalysis by these enzymes relies on a complicated reaction mechanism primarily involving the delivery of electrons from a di-flavin, redox partner (the NADPH cytochrome P450 reductase (CPR)). One of the deleterious consequences of P450-mediated catalysis is that harmful reactive oxygen species (e.g. superoxide and hydrogen peroxide) are formed as a by-product of xenobiotic metabolism. An interesting aspect of P450-mediated metabolism in the liver is that the ratio of CPR:P450 expression ranges from 1:5 to 1:20. Thus, it would seem that most xenobiotic metabolism would be extremely limited by the inability of the P450 enzymes to totally interact with CPR. However, evidence now suggests that the P450 enzymes function as homo- and hetero-aggregates which have different catalytic activities depending on the P450 enzymes present in the mixed complexes. Much of my research is devoted to characterizing the functional consequences of P450 aggregation by examining the tendency of different P450s to form mixed complexes and determining the effects that that these associations have on the catalytic activities of the individual enzymes.
Our lab also studies another membrane-bound enzyme expressed in the endoplasmic reticulum, the heme oxygenase-1 (HO-1). HO-1 uses molecular oxygen and electrons from CPR to convert heme to carbon monoxide, ferrous heme, and biliverdin. This is an important cellular function as free heme is lipid soluble and has been shown to facilitate the formation of highly destructive, reactive oxygen species. HO-1 has been shown to be cytoprotective and is highly inducible by a wide variety of different types of environmental stress. This role has largely been attributed to the antioxidant effects of heme metabolism. In collaboration with the laboratory of Dr. Wayne Backes, which has, for the first time, successfully developed an expression system for the full-length HO-1, we are testing the ability of HO-1 to function as an antioxidant by regulating the activity of the P450 system. We believe this role is likely given that both P450 and HO-1 probably compete for the limiting supply of CPR in the liver endoplasmic reticulum, and HO-1 metabolizes the heme prosthetic group necessary for P450 catalysis. This putative function makes sense intuitively because regulation of P450 activity by HO-1 would be related to the level of HO-1 induction by environmental stress. At this stage, this arm of research is focused on examining the relative abilities of HO-1 and different enzymes of P450 to bind CPR. In this regard we are looking at the ability of HO-1 to influence both the metabolism of xenobiotics and the generation of reactive oxygen species by P450. With both areas of study, we are determining the conditions that influence associations of HO-1, P450, and CPR by using various techniques (e.g. chemical cross-linking, immunoprecipitation, and BRET (bioluminescence resonance energy transfer)).
1. Reed, J.R.; Cawley, G.F.; and Backes, W.L. (2010) Inhibition of cytochrome P450 1A2-mediated metabolism and production of reactive oxygen species by heme oxygenase-1 in rat liver microsomes. Drug Metab. Lett. (In Press).
2. Reed, J.R.; Eyer, M.; and Backes, W.L. (2010) Functional interactions between cytochromes P450 1A2 and 2B4 require both enzymes to reside in the same phospholipid vesicle: evidence for physical complex formation. Jour. Biol. Chem. 285, 8942-8952.
3. Reed, J.R.; Huber, W.J.; and Backes, W.L. (2010) Human heme oxygenase-1 efficiently catabolizes heme in the absence of biliverdin reductase. Drug Metab. Dispos. 38, 2060-2066.
4. Reed, J.R.; Brignac-Huber, L.M.; and Backes, W.L. (2008) Physical incorporation of NADPH-cytochrome P450 reductase and cytochrome P450 into phospholipid vesicles using glycocholate and Bio-Beads. Drug Metab. Dispos. 36, 582-588.
5. Huber, W.J.; Marohnic, C.C.; Peters, M.; Alam, J.; Reed, J.R.; Masters, B.S.S.; and Backes, W.L. (2009) Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system. Drug Metab. Dispos. 37, 857-864.
6. 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.
7. 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.
8. 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 2B4 and P450 reductase. J. Inorg. Biochem.97, 265-75.