Cytochromes P450 (P450s or CYPs) are a
superfamily of
enzymes containing
heme as a
cofactor that mostly, but not exclusively, function as
monooxygenases.[1] However, they are not omnipresent; for example, they have not been found in Escherichia coli.[2] In mammals, these enzymes oxidize
steroids,
fatty acids,
xenobiotics, and participate in many biosyntheses.[1] By hydroxylation, CYP450 enzymes convert xenobiotics into hydrophilic derivatives, which are more readily excreted.
Genes encoding P450 enzymes, and the enzymes themselves, are designated with the
root symbolCYP for the
superfamily, followed by a number indicating the
gene family, a capital letter indicating the subfamily, and another numeral for the individual gene. The convention is to italicize the name when referring to the gene. For example, CYP2E1 is the gene that encodes the enzyme
CYP2E1—one of the enzymes involved in
paracetamol (acetaminophen) metabolism. The CYP nomenclature is the official naming convention, although occasionally CYP450 or CYP450 is used synonymously. These names should never be used as according to the nomenclature convention (as they denote a P450 in family number 450). However, some gene or enzyme names for P450s are also referred to by historical names (e.g. P450BM3 for CYP102A1) or functional names, denoting the catalytic activity and the name of the compound used as substrate. Examples include
CYP5A1,
thromboxane A2 synthase, abbreviated to
TBXAS1 (ThromBoXane A2Synthase 1), and
CYP51A1, lanosterol 14-α-demethylase, sometimes unofficially abbreviated to LDM according to its substrate (Lanosterol) and activity (DeMethylation).[3]
The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of one atom of oxygen into the aliphatic position of an organic substrate (RH), while the other oxygen atom is
reduced to water:
RH + O2 + NADPH + H+ → ROH + H2O + NADP+
Related hydroxylation enzymes
Many
hydroxylation reactions (insertion of
hydroxyl groups) use CYP enzymes, but many other hydroxylases exist.
Alpha-ketoglutarate-dependent hydroxylases also rely on an Fe=O intermediate but lack hemes. Methane monooxygenase, which converts methane to methanol, are non-heme iron-and iron-copper-based enzymes.[7]
Mechanism
Structure
The active site of cytochrome P450 contains a heme-iron center. The iron is tethered to the protein via a
cysteinethiolateligand. This cysteine and several flanking residues are highly conserved in known P450s, and have the formal
PROSITE signature consensus pattern [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C - [LIVMFAP] - [GAD].[8] In general, the P450 catalytic cycle proceeds as follows:
Catalytic cycle
Substrate binds in proximity to the
heme group, on the side opposite to the axial thiolate. Substrate binding induces a change in the conformation of the active site, often displacing a water molecule from the distal axial coordination position of the heme iron,[9] and changing the state of the heme iron from low-spin to high-spin.[10]
Substrate binding induces electron transfer from NAD(P)H via
cytochrome P450 reductase or another associated
reductase,[11] converting Fe(III) to Fe(II).
Molecular oxygen binds to the resulting ferrous heme center at the distal axial coordination position, initially giving a
dioxygen adduct similar to oxy-myoglobin.
The peroxo group formed in step 4 is rapidly protonated twice, releasing one molecule of water and forming the highly reactive species referred to as P450 Compound 1 (or just Compound I). This highly reactive intermediate was isolated in 2010,[12] P450 Compound 1 is an iron(IV) oxo (or
ferryl) species with an additional oxidizing equivalent
delocalized over the
porphyrin and thiolate ligands. Evidence for the alternative perferryl
iron(V)-oxo[9] is lacking.[12]
Depending on the substrate and enzyme involved, P450 enzymes can catalyze any of a wide variety of reactions. A hypothetical hydroxylation is illustrated. After the hydroxylated product has been released from the active site, the enzyme returns to its original state, with a water molecule returning to occupy the distal coordination position of the iron nucleus.
An alternative route for mono-oxygenation is via the "peroxide shunt" (path "S" in figure). This pathway entails oxidation of the ferric-substrate complex with oxygen-atom donors such as peroxides and hypochlorites.[13] A hypothetical peroxide "XOOH" is shown in the diagram.
Mechanistic details, including the
oxygen rebound mechanism, have been investigated with synthetic analogues, consisting of iron oxo heme complexes.[14]
Spectroscopy
Binding of substrate is reflected in the spectral properties of the enzyme, with an increase in absorbance at 390 nm and a decrease at 420 nm. This can be measured by difference spectroscopies and is referred to as the "type I" difference spectrum (see inset graph in figure). Some substrates cause an opposite change in spectral properties, a "reverse type I" spectrum, by processes that are as yet unclear. Inhibitors and certain substrates that bind directly to the heme iron give rise to the type II difference spectrum, with a maximum at 430 nm and a minimum at 390 nm (see inset graph in figure). If no reducing equivalents are available, this complex may remain stable, allowing the degree of binding to be determined from absorbance measurements in vitro[13]
C: If carbon monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum with a maximum at 450 nm. However, the interruptive and inhibitory effects of CO varies upon different CYPs such that the CYP3A family is relatively less affected.[15][16]
Estabrook RW (December 2003). "A passion for P450s (Remembrances of the early history of research on cytochrome P450)". Drug Metabolism and Disposition. 31 (12): 1461–1473.
doi:
10.1124/dmd.31.12.1461.
PMID14625342.
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^Danielson PB (December 2002). "The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans". Current Drug Metabolism. 3 (6): 561–597.
doi:
10.2174/1389200023337054.
PMID12369887.
^Nelson DR (January 2011). "Progress in tracing the evolutionary paths of cytochrome P450". Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1814 (1): 14–18.
doi:
10.1016/j.bbapap.2010.08.008.
PMID20736090.
^Smith AT, Pazicni S, Marvin KA, Stevens DJ, Paulsen KM, Burstyn JN (April 2015). "Functional divergence of heme-thiolate proteins: a classification based on spectroscopic attributes". Chemical Reviews. 115 (7): 2532–2558.
doi:
10.1021/cr500056m.
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