Abstract
This paper by Alfred G. Hildebrandt and Ronald W. Estabrook at the University of Texas (Southwestern) Medical School, led to the concept of cytochrome b5 (b5) as an auxiliary protein facilitating some cytochrome P450 (P450) reactions in the liver and other tissues. The gist of the paper is that DPNH (now known as NADH) enhanced rates of TPNH (now NADPH)-supported N-demethylation of O-ethylmorphine in rat liver microsomes. The conclusion was that b5 was providing an electron to the ferrous-oxy form of P450 (Fe2+O2), which was supported by some spectral observations on the oxidation state of b5 in the microsomes in the steady state. This observation led to a flurry of activity, which is still in progress. This paper has been cited 678 times in Google (558 in Clarivate), and I have often cited it myself. A PubMed search for the terms P450 and b5 yielded 2,244 results.
This paper [1] by Alfred G. Hildebrandt, a postdoc/junior faculty, and his mentor Ronald W. Estabrook at the University of Texas (Southwestern) Medical School, led to the concept of cytochrome b5 (b5) as an auxiliary protein facilitating some cytochrome P450 (P450) reactions in the liver and other tissues. The gist of the paper is that DPNH (now known as NADH) enhanced rates of TPNH (now NADPH)-supported N-demethylation of O-ethylmorphine in rat liver microsomes. The conclusion was that b5 was providing an electron to the ferrous-oxy form of P450 (Fe2+O2), which was supported by some spectral observations on the oxidation state of b5 in the microsomes in the steady state. This observation led to a flurry of activity, which is still in progress. This paper has been cited 678 times in Google (558 in Clarivate), and I have often cited it myself. A PubMed search for the terms P450 and b5 yielded 2,244 results.
What do we know today? The rat P450s involved were probably 2B1, 2C6, and 2C11. Later the b5 stimulation could be shown with adrenal P450 17A1 steroid lyase activity, which is the most dramatic case [2].
Like many scientific phenomena, what started out as a simple explanation of it became more complex. With individual purified P450 enzymes, b5 could either stimulate, inhibit, or have no effect [3]. Using rapid kinetic experiments, Waskell’s group could show that electron transfer from ferrous b5 to P450 2B4 occurred in a productive manner in cyclohexane hydroxylation d-benzphetamine N-demethylation [4].
However, experiments with apo-b5, devoid of heme and incapable of electron transfer, showed the same stimulation of the activities of human P450 3A4 [5] and 17A1 [6]. A more comprehensive study of multiple human P450s showed some that require the heme of b5 (2E1) for stimulation but others do not (2A6, 2B6, 2C8, 2C9, 2C19, 3A4, 3A5, 17A1) [7]. The most likely explanations for b5 stimulation are direct electron transfer (to a Fe2+O2 complex) and an allosteric function, and either mechanism could be coupled with reduced waste of electrons in generation of H2O2 [8]. Apo-b5 has also been shown to be effective in stimulating P450 17A1 steroid lyase activity in cells [9]. Lack of b5 binding to P450 17A1 appears to be the cause of androgen insufficiency in clinical cases and can be demonstrated in vitro [10, 11]. Although the redox potential of b5 in unfavorable for providing the first electron to reduce ferric P450, NADH-dependent P450 reactions have been demonstrated with NADH-b5 reductase and b5 [12]. Further, this appears to be very feasible in mice in which liver is devoid of NADPH-P450 reductase due to a selective knockout [13].
The safest conclusion is that b5 can influence P450 reactions through multiple mechanisms. Hildebrandt and Estabrook really started it all, even if the questions about mechanisms persist. There is certainly room for more insight into the phenomena.
Prof. Ronald W. Estabrook died in 2013 after a long and distinguished academic career at the University of Pennsylvania and University of Texas. Prof. Alfred G. Hildebrandt returned to Germany in 1970 and had a career at the Free University of Berlin and then in the Federal Regulatory Institute for Consumer Protection and the Drug Institute at the Federal Institute for Drugs and Medical Devices in Germany. He retired in 2002 and is living in Bonn, at the age of 85.
References
- [1].Hildebrandt A, Estabrook RW, Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactions, Arch. Biochem. Biophys 143 (1971) 66–79 [DOI] [PubMed] [Google Scholar]
- [2].Katagiri M, Suhara K, Shiroo M, Fujimura Y, Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17,20-lyase reaction, Biochem. Biophys. Res. Commun 108 (1982) 379–384 [DOI] [PubMed] [Google Scholar]
- [3].Gorsky LD, Coon MJ, Effects of conditions for reconstitution with cytochrome b5 on the formation of products in cytochrome P-450-catalyzed reactions, Drug Metab. Dispos 14 (1986) 89–96 [PubMed] [Google Scholar]
- [4].Zhang H, Im SC, Waskell L, Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4, J. Biol. Chem 282(41) (2007) 29766–29776 [DOI] [PubMed] [Google Scholar]
- [5].Yamazaki H, Johnson WW, Ueng YF, Shimada T, Guengerich FP, Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5, J. Biol. Chem 271(44) (1996) 27438–27444 [DOI] [PubMed] [Google Scholar]
- [6].Auchus RJ, Lee TC, Miller WL, Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer, J. Biol. Chem 273 (1998) 3158–3165 [DOI] [PubMed] [Google Scholar]
- [7].Yamazaki H, Nakamura M, Komatsu T, Ohyama K, Hatanaka N, Asahi S, Shimada N, Guengerich FP, Shimada T, Nakajima M, Yokoi T, Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli, Prot. Express. Purif 24(3) (2002) 329–337. 10.1006/prep.2001.1578 [DOI] [PubMed] [Google Scholar]
- [8].Peng HM, Im SC, Pearl NM, Turcu AF, Rege J, Waskell L, Auchus RJ, Cytochrome b5 activates the 17,20-lyase activity of human cytochrome P450 17A1 by increasing the coupling of NADPH consumption to androgen production, Biochemistry 55(31) (2016) 4356–4365. 10.1021/acs.biochem.6b00532 [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Storbeck KH, Swart AC, Lombard N, Adriaanse CV, Swart P, Cytochrome b5 forms homomeric complexes in living cells, J. Steroid Biochem. Mol. Biol 132(3-5) (2012) 311–321. 10.1016/j.jsbmb.2012.07.006 [DOI] [PubMed] [Google Scholar]
- [10].Peng HM, Liu J, Forsberg SE, Tran HT, Anderson SM, Auchus RJ, Catalytically relevant electrostatic interactions of cytochrome P450c17 (CYP17A1) and cytochrome b5, J. Biol. Chem 289(49) (2014) 33838–33849. 10.1074/jbc.M114.608919 [DOI] [PMC free article] [PubMed] [Google Scholar]
- [11].Kim D, Kim V, McCarty KD, Guengerich FP, Tight binding of cytochrome b5 to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis, J. Biol. Chem 296 (2021) 100571. 10.1016/j.jbc.2021.100571 [DOI] [PMC free article] [PubMed] [Google Scholar]
- [12].West SB, Levin W, Ryan D, Vore M, Lu AYH, Liver microsomal electron transport systems. II. The involvement of cytochrome b5 in the NADH-dependent hydroxylation of 3,4-benzpyrene by a reconstituted cytochrome P-488-containing system., Biochem. Biophys. Res. Commun 58 (1974) 516–522 [DOI] [PubMed] [Google Scholar]
- [13].Henderson CJ, McLaughlin LA, Wolf CR, Evidence that cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the hepatic cytochrome P450 systems, Mol. Pharmacol 83(6) (2013) 1209–1217. 10.1124/mol.112.084616 [DOI] [PubMed] [Google Scholar]