Cytochrome P450 Is Present in Both Ferrous and Ferric Forms in the Resting State within Intact Escherichia coli and Hepatocytes

Johnston, Wayne A.; Hunter, Dominic J. B.; Noble, Christy; Hanson, Graeme R.; Stok, Jeanette E.; Hayes, Martin A.; Voss, James J. De; Gillam, Elizabeth M. J.

doi:10.1074/jbc.m111.300871

Cited by 30 publications

(19 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The first step in the reaction cycle is generally agreed to be substrate binding, in that the presence of the substrate facilitates the introduction of an electron to the ferric iron in some but not all cases (6). Binding of substrates can also occur after initial iron reduction (7,8).…”

mentioning

confidence: 99%

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Guengerich

Wilkey

Phan³

2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.

show abstract

mentioning

confidence: 99%

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Guengerich

Wilkey

Phan³

2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This is likely due to inefficient reduction of Fe(III) to the active Fe(II) catalyst for carbene transfer, as the redox potential of NAD + /NADH (E°′ = −320 mV; all potentials versus standard hydrogen electrode) is less negative than that of low spin Fe(III)/Fe(II) in P450-BM3 (E°′ = −430 mV). Because P450s exist in both the ferric and ferrous forms in intact Escherichia coli cells, 12 we posited that catalysis could also be achieved using intact cells. Addition of EDA and aniline to cells expressing H2-5-F10 provided 1 in 38% yield.…”

Section: Resultsmentioning

confidence: 99%

Cytochrome P450-catalyzed insertion of carbenoids into N–H bonds

et al. 2014

View full text Add to dashboard Cite

Expanding nature's catalytic repertoire to include reactions important in synthetic chemistry will open new opportunities for ‘green’ chemistry and biosynthesis. We demonstrate enzyme-catalyzed insertion of carbenoids into N-H bonds. This type of bond disconnection, which has no counterpart in nature, can be mediated by variants of the cytochrome P450 from Bacillus megaterium. The N-H insertion reaction takes place in water, provides the desired products in 26-83% yield, forms the single addition product exclusively, and does not require slow addition of the diazo component

show abstract

“…The binding of a substrate that also displaces the axial water ligand (Scheme 1, state A to B ) induces a change in the iron atom from a low-spin to a high-spin state [9]. In the presence of the substrate, the absorption maximum of the Soret band shifts to a shorter wavelength from spectrum [ iv ] at 418 nm (Fig.…”

Section: Steady-state Enzyme Kinetics Versus Rapid Kineticsmentioning

confidence: 99%

Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms

Yoshimoto

Auchus

2016

The Journal of Steroid Biochemistry and Molecular Biology

View full text Add to dashboard Cite

All cytochrome P450 enzyme reactions involve a catalytic cycle with several discreet physical or chemical steps. This cycle ends with the formation of the reactive heme iron-oxygen complex, which oxygenates substrate. While the steps might be very similar for each P450 enzyme, the rates of each step varies tremendously for each enzyme and sometimes even for different reactions catalyzed by the same enzyme. For example, the rate-limiting step for most bacterial P450 enzymes, with turnover numbers over 1,000 sec−1, is the second electron transfer. In contrast, steroidogenic P450s from eukaryotes catalyze much slower reactions, with turnover numbers of ~5–250 min−1; therefore, assumptions about kinetic properties for the mammalian P450 enzymes based on the bacterial enzymes are tenuous. In order to dissect the rates for individual steps, special techniques that isolate individual steps and/or single turnovers are required. This article will review the theoretical principles and practical considerations for several of these techniques, with illustrative published examples. The reader should gain an appreciation for the appropriate methods used to interrogate particular steps in the P450 reaction cycle.

show abstract

Cytochrome P450 Is Present in Both Ferrous and Ferric Forms in the Resting State within Intact Escherichia coli and Hepatocytes

Cited by 30 publications

References 44 publications

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Cytochrome P450-catalyzed insertion of carbenoids into N–H bonds

Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms

Contact Info

Product

Resources

About