Cytochrome P450:

Structure and Function.

Introduction:

Cytochrome P450 is one of the most studied enzyme up to now since 1958.
Presently Cytochrome P450 is a collection of various derivative forms that is further
organized to family and subfamily based on their amino acid sequence and known
physiological function. So far there are about 200 families of Cytochrome P450
discovered whereby 80 families are from bacteria, 50 families from plant, 40 families
from animals and 25 families are from fungi. In mammals apparently there are 50-80
genes that are responsible for Cytochrome P450 expression, including plants the P450
are expressed at specific tissues and at specific developmental stage indicating diverse
metabolic roles.

Structure:

As a hemoprotein,
Cytochrome P450 also has a
protoheme with the 5th ligand
contributing to a characteristic
absorption at 450nm as cytochrome
P450 becomes reduced. P450 can
exist in two forms either water
soluble in prokaryotes or
membrane bound especially in
mitochondria and Endoplasmic
Reticulum in Eukaryotic organisms
respectively. In principle,
Cytochrome P450 is made up of
12α helices and 5 antiparallel β
sheets with the heme located
between Helix I & Helix L and at
the bottom of an internal pocket
which in turn is surrounded by
hydrophobic amino acid residues Fig. 1: Fold & secondary structure content of the 3 P450s. The
(Fig. 1). In overall this gives molecules are viewed from the substrate access (distal) face. α-Helices
Cytochrome P450 an asymmetrical are presented as purple coils, 310-helices and π-helices as green coils,
β-strands as red arrows, and loops as yellow tubes. The heme
triangular prism shape irrespective molecules are shown as ball-and-stick models. The visible secondary
whether it is eukaryotic or structures of each molecule are labeled (α-helices A, B', D-G, I and L,
and β-sheets β1-β5), as are the amino (N) and carboxyl (C) termini.
prokaryotic. Since eukaryotic (Hasemann C.A., 1995)
cytochrome P450 are membrane
bound, a specific sequence called
‘signal anchor sequence’ are present which are highly hydrophobic and consists of 20-
25 amino acids at the amino-terminus of microsomal P450. On the other hand the
presequence of mitochondrial P450 is 20-40 amino acids long, providing water
soluble properties in the cytoplasm after being synthesized and is then proteolytically
removed after entering into the mitochondria. Non the less, both forms of P450 are
500 amino acids long in their primary structure and hence the presequence of
mitochondrial P450 are more or less equivalent to the signal anchor sequence of the
microsomal P450.

Function:

It was stipulated that the primitive function of cytochrome P450 was to

synthesize and degrade sterols necessary for maintenance of the plasma membrane.
As the eukaryotic organisms evolve, other P450 forms were created from primitive
P450 hydrophobic substrate socket. Cytochrome P450s are mainly involved in
monooxygenase reactions besides catalyzing few ‘intramolecular oxygen atom
transfer’. Main reaction of mono-oxygenation involves multitude of hydrophobic
substrates, requiring molecular oxygen and reducing agents NADPH or NADH in
eukaryotic and prokaryotic organisms respectively. Generally in eukaryotic
organisms, the mitochondrial P450 is assisted by 2 soluble protein components that
catalyze electron transfer from NADPH to P450. Similarly there are also 2
components in microsomal P450 however these are also membrane bound. The
mechanism for cytochrome P450-catalyzed oxygenation reaction is called as ‘cyclic
reaction mechanism’. This mechanism is basically made up of a number of steps;
Heme of P450 substrate bound complex is reduced by the transfer of electron and
another Heme-oxygen bound molecule is also reduced by the transfer of electron,
splitting the O-O bond. Consequently one of the oxygen atom is reduce to water while
the other is transferred to the substrate molecule and also superoxide anion by product
of the oxygenation reaction is produced. The site of oxygenation on the substrate
depends on the topology of the substrate during binding relative to the activated
oxygen atom of the heme. In the case of the intramolecular oxygen atom transfer, this
involves the rearrangement of substrate molecule by splitting an endoperoxide bond.

In the synthesis of steroid hormones, the oxygenase reactions are mainly

hydroxylation reactions. Six P450s i.e. three microsomal and three mitochondrial
have been found to participate alongside two hydroxysteroid dehydrogenases in
adrenocortical and gonadal steroid hormone biosynthesis. The expression of tissue
specific steroidogenic P450s also depends on Ad4BP transcription factor or SF-1
steroid hormone/thyroid hormone receptor. Steroidogenic P450’s could also be
expressed in non-steroidogenic tissues presumably converting incoming steroid
hormones into other types of steroid hormones. Further metabolism of steroids from
the blood occurs in the liver by P450 catalyzed hydroxylation reactions followed by
other conjugation enzymes to inactivate and excrete them out from the body.
Table 1: Example of P450 forms and functions.
Cytochrome P450 Function
scc, c17, c21,
Biosynthesis of adrenocortical & gonadal steroid hormones
arom, 11β, aldo
14DM (51A) Biosynthesis of sterols (cholesterol, ergosterol, phytosterols)
c7α (7A1) Catabolism of cholesterol to bile acid
c27 (27A1) Mitochondrial P450 catalyzing 25-hydroxylation of vitamin D3 in the kidney
c1α (27B1) Mitochondrial P450 catalyzing 1α-hydroxylation of 25-hydroxyvitamin D3 in liver
Catalyze 24-hydroxylation of 25-hydroxyvitamin D3 to inactivate 24,25-
c24 (24A1)
dihydroxyvitamin D3
c24 Catalyze monooxgenation of 1,25-hydroxyvitamin D3 to calcitroic acid.
RA, RAI (26A) Catalyze the metabolism of retinoic acid & regulate its conc. in tissue cells.
1A1 3MC xenobiotic induced microsomal cytochrome metabolism
2D6 Debrisoquine/sparteine drug oxidation
2B1, 2B2 PB drug metabolism

Another example of P450 catalyzed reaction is in the biosynthesis of sterols

(cholesterol, ergosterol, phytosterols) whereby 14α-methyl group is oxidatively
removed from sterols intermediates (lanosterol, obtusiferiol). Catabolism of sterol
(cholesterol) into bile acid initially by 7α-hydroxylation is also catalyzed by P450.
Moreover P450 is also involved in ω-hydroxylation of fatty acids. For example
eicosatetraenoic acids (HETEs) are formed via ω- & ω-1-hydroxlation while
epoxyeicosatetraenoic acids (EETs) is via epoxidation of double bond of arachidonic
acid by microsomal P450 in the kidney. Other examples are the ω-hydroxylation of
prostaglandins and leukotrienes by microsomal Family IV P450, the synthesis &
metabolism of eicosanoids, conversion of Vitamin D3 to 1,2,5-dihydroxyvitamin D3
(active form) and consequently to calcitroic acid, and the catalysis of retinoic acid.

It was found that P450 could either be under hormonal control or be induced
by certain chemical compounds i.e. Xenobiotics. The later particularly those P450 in
Family I & II, detoxify and defend against low molecular weight foreign compounds
avoiding its accumulation in the body by metabolizing hydrophobic chemicals firstly
into more polar and then hydrophilic compounds to enable further enzyme attacks. An
example is TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) a strong genotoxic
compound. As the compound binds to AhR Receptor, this complex moves into the
nucleus and forms heterodimer with Arnt (AhR translocator). This complex then
binds to XRE sequence (xenobiotics responsive element) i.e. an expression enhancer
& regulator of CYP1A1 gene expression that codes for P4501A1 which is aryl
hydrocarbon hydroxylase. Despite its initial function of P450 to protecting the
organism from being intoxicated, unfortunately metabolism of some compounds such
as benzo(a)pyrene and other PAH (polyaromatic hydrocarbons) results in the
production of metabolites e.g. epoxides which are highly reactive to proteins and
nucleic acids, which leads to cytotoxicity and genotoxicity of the organism.

References:
Omura T. (1999). Forty Years of Cytochrome P450. Biochemical and Biophysical
Research Communications, 266, 680-698.

Hasemann C.A. Kurumbail R.G, Boddupalli S.S., Peterson J.A. & Deisenhofer J.
(1995). Structure and function of cytochromes P450: a comparative analysis of three
crystal structures. Structure, 2:41-62.