Cytochrome P450

• Heme protein• Terminal oxidase of the mixed-function

oxidase (MFO) electron-transfer system• Located in the smooth endoplasmic

reticulum of all major organs and tissues• Uses NADPH as a source of reducing

equivalents• Inducible

Overall reaction

R-H + O2 + NADPH + H+

R-OH + H2O + NADP+

Ferric protoporphyrin IX

N

N-

N

-N

Fe+3

CH3 HCCH2

H3C

CH3

CH3

CH

CH2

CH2

H2CC

O

OH

H2C

H2CC

O

OH

Protoporphyrin IX

Catalytic cycle of cytochrome P450

from NADPH-cytC reductase

Fe3+

Fe3+-RH

Fe2+-RH

O2

[Fe2+-RH]

HO2-

[Fe2+-RH]

HO22-

Fe3+-RH

+ RH

+ e-

+O2

ROH

H+ + e-

NADPH

NADH

H2O

H2O2H+

H+

O2-.

P450 and reductase in endoplasmic reticulum

The P450 gene superfamily• Format of nomenclature:

CYPFamily/Subfamily/Gene• Family = 1, 2, …150 and counting

– ~40% aa similarity• Subfamily = A, B,…H…

– 55-65% aa similarity• Gene = 1, 2..10 or above

– >97% aa similarity (allelic variants)• Families grouped in Clans

Sub-Family family Gene

CYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylation

CYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation

B 1 Aliphatic hydroxylation2 O-deethylation

C 1 - 20+2C19, mephenytoin hydroxylase

Demethylation

Deethylation

O

N

H2C CH3

H C CH3

O

OH

NH C CH3

O

+

Phenacetin Acetaminophen

C CH3

o

H

Acetaldehyde

OCH3

OH

COH

H

+

Anisole Phenol Formaldehyde

Sub-Family family Gene

CYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylation

CYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation

B 1 Aliphatic hydroxylation2 O-deethylation

C 1 - 20+2C19, mephenytoin hydroxylase

Sub- Family family Gene

CYP2 D 1 - 6+2D6, debrisoquine hydroxylase

E 1 C- and N-hydroxylationsmall molecules

2 F 1

CYP3 A 1-43A4

CYP4 A 1 Lauric acid - and -1 hydroxylation

Sub-Family family GeneCYP11 (mito) A 1 Steroid 11-hydroxylation

CYP17 A 1 Steroid 17-hydroxylationCYP21 A 1 Steroid 21-hydroxylation

CYP51 A 1 (Plants, yeast)CYP52-66 A Yeasts, fungiCYP71-99, 701 PlantsCYP101 A 1 Pseudomonas putida P450cam

CYP102-132 A Bacteria

Induction

• Increased transcription• Increased protein synthesis• Enhanced stability of protein• Synthesis of enzyme with higher catalytic

activity

Example:Ah-locus mediated induction

AhR, receptor in cytoplasm, binds ligand: eg PAHs, TCDD, some PCBs

Bound AhR loses 2 heat-shock proteins (hsp90), becomes phosphorylated

Activated bound AhR migrates to nucleus, forms complex with Ah receptor nuclear translocation factor Arnt

AhR-Arnt complex binds to regulatory sequences in DNA (DRE, dioxin-responsive elements)

Transcription of CYP1A1 gene and other genes

Other “inducers” also interact with receptors

• CAR, responds to phenobarbital-type inducers, regulates CYP2B, CYP3A4, CYP reductase, transferases (?)

• PXR, CYP3A• PPARα, CYP4A• LXR, FXR control enzymes involved in

bile acid and lipid metabolism