Rend. Fis. Acc.

Lincei
DOI 10.1007/s12210-016-0565-z

CONCEPTS IN CATALYSIS

Heme iron centers in cytochrome P450: structure and catalytic

activity
Gianfranco Gilardi1 • Giovanna Di Nardo1

Received: 19 July 2016 / Accepted: 11 August 2016

Ó Accademia Nazionale dei Lincei 2016

Abstract Heme iron centers are found in a wide range of exploit them for biotechnological applications, but careful
proteins where they play different roles for many crucial attention must be paid in not altering their delicate struc-
biological processes, including catalysis. Among heme- ture on which their correct function strictly depends.
containing enzymes, the cytochromes P450 superfamily
comprises members distributed in all domains of life where Keywords Cytochromes P450  Heme reactivity 
they participate in the metabolism of endogenous and Monooxygenases
exogenous compounds. These enzymes can perform a
series of oxidative reactions on a broad range of chemically
different substrates and for this reason are optimal candi-
dates for biocatalytic purposes and, in general, technolog- 1 Introduction
ical applications. In this review, the general features of
these enzymes will be discussed with particular emphasis Enzymes are attractive catalysts able to combine the ability
on the structural insights obtained through X-ray crystal- to carry out stereo- and regio-specific reactions at high
lography to understand the key steps of their catalytic yield with the capacity to perform in environmentally
mechanism where oxygen is activated. Moreover, one of sustainable mild conditions. Due to the characteristics of
the finest multi-step reactions catalyzed by the cytochrome the cellular environment in which nature designed them to
P450 aromatase, dealing with the conversion of androgens function, they often function in aqueous solutions at neutral
into estrogens, will be discussed in details. The X-ray pH values and mild temperatures. Despite these attractive
structure of this enzyme, together with site-directed properties, the applicability of enzymes in industrial pro-
mutagenesis experiments has elucidated the role of key cesses is often limited by their complex and unstable 3D
residues involved in substrate binding and catalysis. This structure that is designed to work in the living environment
last example shows how the function and structure in for a set length of time to ensure degradation and
cytochromes P450 are closely inter-correlated to achieve a replacement depending on the metabolic needs of the cell.
complex finely tuned catalytic mechanism. It is possible to Protein engineering, with its rational design or random
mutagenesis approaches, can help in producing enzymes
more amenable to industry. Here, durable and robust
This contribution is the written, peer-reviewed version of a paper molecules are needed able to withstand the test of time,
presented by a participant to the Conference ‘‘Concepts in catalysis: often operating in extreme conditions.
from heterogeneous to homogeneous and enzymatic catalysis’’ held at To perform their activity, enzymes often require cofac-
Accademia dei Lincei in Rome on February 25–26, 2016.
tors or prosthetic groups inserted in an active site with an
& Gianfranco Gilardi architecture defined by amino acids with side chains con-
[email protected] ferring specific shapes, sizes, charges and reactivity. This
1
forms the basis on which their substrate chemo-, regio- and
Department of Life Sciences and Systems Biology,
University of Torino, Via Accademia Albertina 13,
stereo-selectivity is based as well as the variety of reactions
10123 Turin, Italy that they can catalyze.

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In the orthodox view, enzymes are considered as linked to the porphyrin ring and the way the cofactor is
homogeneous catalysts, because they work in the same bound to the protein matrix, different heme types have
phase of their substrates and products. However, if neces- been classified and have been associated with specific
sary they can be easily immobilized on surfaces and functions. The b-type heme (heme-b) is present for
therefore be separated, owing to their much larger dimen- example in proteins involved in O2 storage and transport
sions. This last point can be an issue when working with (myoglobin and hemoglobin) as well as in O2-mediated
homogeneous catalysis. It can therefore be argued that catalysis (peroxidase, catalase, cytochromes P450). Heme-
enzymes represent a class of catalysts in their own right, a is present in those enzymes reducing O2 to H2O during
due to their unique and complex nature where a homoge- catalysis (cytochrome c oxidase) and heme-c is present in
neous catalytic site is included in the protein matrix that electron transfer proteins such as cytochrome c. From the
plays an active role in promoting the formation of specific catalytic point of view, in the presence of heme and O2,
interactions between the catalytic center and the substrate. different physiological phenomena can take place: for
Moreover, the protein fold has a crucial role in the selec- example, energy can be captured (cytochrome c oxidase)
tion and recruitment of the substrate from the surface to the or useful metabolites and hormones can be produced.
catalytic site through specific channels that can be different Heme catalysts are also involved in the detoxification of
from those used by the reaction products to be released by the organism from xenobiotic compounds as well as
the enzyme. The ligands usually required to develop a reactive oxygen species that can be produced by other
homogeneous catalyst are already present in the enzymes heme-containing catalysts.
and are represented by the amino acids that line the cat- In this short review, we will focus on a superfamily
alytic cleft conferring selectivity. In this view, enzymes of heme catalysts involved in many of these processes,
combine some advantages of both homogeneous and depending on the organism and the tissue where they
heterogeneous catalysis. are found, but also the conditions where the organism
As enzymes are normally very selective, the mainte- lives.
nance of their 3D structure is crucial, and this is
achieved in a quite narrow range of conditions. Such
conditions are usually mild and therefore the use of high
temperatures or strong acids and bases can usually be 3 Cytochromes P450 as biocatalysts
avoided when using a biocatalyst with an advantage in
terms of energy costs and environmental sustainability. Cytochromes P450 are a superfamily of heme-containing
On the other hand, if required, enzymes can be extracted enzymes involved in both anabolic and catabolic pathways.
from organisms adapted to survive in extreme conditions They are found in all domains of life, including viruses
such high temperatures or extreme pH. Alternatively, (Sigel et al. 2007; Lamb et al. 2009), where they play
advances in molecular and structural biology techniques crucial roles in the metabolism of both endogenous and
have provided the opportunity to manipulate enzymes exogenous compounds. In mammals, for example, they are
with the aim of producing a biocatalyst optimized for a involved in steroid hormones, vitamin D and terpenoids
specific reaction. In particular, protein engineering can biosynthesis as well as detoxifications of xenobiotics
be used to change the amino acid identity in specific including drugs (Ortiz de Montellano 2015). In plants and
positions at the DNA level with an effect on the struc- insects, they are also involved in chemical defense mech-
tural features of the enzyme. This approach has been anisms, including herbicide and insecticide resistance
widely used to increase the enzyme performance, the (Schuler and Werck-Reichhart 2003; Mizutani and Sato
chemo-, regio- and stereoselectivity, and also the stabil- 2011). In bacteria, they are the key players in the adapta-
ity of the enzyme (Valetti and Gilardi 2004) and, tion strategies that make bacteria able to use different
nowadays, enzymes are used for industrial applications molecules, including toxic compounds, as fuels for meta-
(Choi et al. 2015). bolism (Urlacher et al. 2004; Girvan and Munro 2016).
The name cytochromes P450 derives from their heme-
dependent spectroscopic behavior showing a typical
2 Heme centers as catalysts in proteins absorption band at 450 nm when reduced and bound to
carbon monoxide (Omura and Sato 1964). In 2016, there
The physico-chemical properties of the iron–porphyrin are 35,166 named P450 sequences in databases, with many
complex have been exploited by nature to develop a wide organisms having different P450 sequences in their gen-
range of proteins working in different cells and subcel- ome, including Homo sapiens that has 57 genes coding for
lular organelles where they perform a wide range of these proteins (Nelson 2009), with the notable exception of
functions. Depending on the nature of the substituents E. coli that has none.

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The chemical diversity of the compounds recognized by 4 The P450 general fold and catalytic cycle
these proteins makes them very attractive in the field of
biocatalysis. Even more attractive is the fact that these X-ray crystallography has been widely used to solve the
proteins catalyze different reactions. They mainly act as crystal structures of many cytochromes P450 from different
monooxygenases carrying out the following reaction: sources. The available structures show that these enzymes
R  H þ O2 þ 2e þ 2Hþ ! R  OH þ H2 O: share a highly conserved fold with a triangular shape,
carrying a small domain rich in b-sheets and a larger
Other oxidative reactions carried out by these enzymes domain formed mainly by a-helices. Even if there are small
include oxidative ester cleavage, dearylation, oxidative differences in the number and location of a-helices and b-
ring coupling, ring formation or expansion, aromatic sheets among different enzymes, most of the secondary
dehalogenation, dearomatization and isomerization (Ortiz structure elements are organized in the same way in the 3D
de Montellano and Nelson 2011; Guengerich 2001). space and heme is located in the core of the protein where
To perform their reactions, cytochromes P450 requires the iron atom is bound to a highly conserved cysteine
two electrons for each catalytic cycle. The electrons are residue. The heme–thiolate ligand motif is protected and
supplied from NADPH to the cytochrome P450 via a the binding of the cofactor to the protein also occurs via
reductase that can be a protein domain included in the other non-covalent interactions such as hydrogen bonds
same polypeptide chain as the catalytic domain or a dif- and electrostatic interactions between the protein arginine
ferent and separate protein. According to the organization residues and the negatively charged heme propionate
of electron transfer partners, cytochromes P450 are clas- groups.
sified into ten classes (Hannemann et al. 2007). Along with The active site of these proteins shows some generally
this classification, most of the mammalian cytochromes conserved features, such as the presence of important water
P450 belong to class II where a cytochrome P450-reduc- molecules in the active site of the protein. Figure 2 shows
tase (CPR) is involved as a redox partner. The two proteins the crystal structure of the heme domain of the bacterial
are linked to the endoplasmic reticulum membrane through cytochrome P450 BM3, a fatty acid monooxygenase (Nahri
a helix that anchors the protein to the membrane. The CPR and Fulco 1986). A water molecule is present as the sixth
carries two cofactors, flavin mononucleotide (FMN) and ligand for the heme iron that is in the low spin state, as
flavin adenine dinucleotide (FAD). Thus, a small redox demonstrated by spectroscopic characterization (Lipscomb
chain is formed where electrons flow from NADPH to 1980; Fisher et al. 1985; Raag and Poulos 1989). More-
FAD, FMN, heme and O2 (Fig. 1). The electron flow is over, this water molecule is part of a network of hydrogen
allowed by a specific protein–protein interaction, based on bonds involving both amino acid groups and other water
the complementarity of two faces of CPR and cytochromes
P450 carrying opposite charges (Fig. 1). Hence, the two
proteins behave like dipoles and, once amino acids of
opposite charge come close, they can interact forming salt
bridges. 2.6 Å

2.9 Å
3.3 Å
e-
2.5 Å
NADPH Thr 268

FAD

DIPOLE

Hinge FMN P450

domain

Fig. 1 Scheme showing the electrostaic interaction between the Fig. 2 Details of the crystal structure of the heme domain of the
membrane-bound mammalian cytochrome P450 reductase (FAD bacterial cytochrome P450 BM3, showing the water molecule (red
domain in orange, hinge domain in green and FMN domain in sphere) sixth ligand to the heme iron (orange). The dotted lines show
yellow) and cytochrome P450 (in red). The arrows indicate the flow the hydrogen bonds involving other water molecules and amino acid
of electrons from NADPH to FAD to FMN to heme groups, including Thr268 key to enzymatic activity

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molecules (Fig. 2). This network involves also the so- entrance of a second proton leads to the release of a water
called oxygen activations motif, a highly conserved region molecule and a highly reactive oxoferryl species is
where a threonine residue is present and involved in the formed, the so-called ‘‘Compound I’’ (Fe4?=O) (step 6 in
hydrogen bond with a catalytically important water Fig. 3). The abstraction of a proton from the substrate
molecule. generates the so-called ‘‘Compound II’’ (Fe4?-OH) which
The catalytic cycle of cytochromes P450 is shown in hydroxylates the substrate radical according to the radical
Fig. 3 and has been studied and elucidated through a rebound mechanism (step 7 in Fig. 3) (Rittle and Green
combination of spectroscopic techniques including X-ray 2010). At this point, the substrate is released as product,
crystallography. and the enzyme returns to the resting state with a water
In the first step (step 1), when the substrate has access molecule in the distal coordination position of the heme
to the active site, in most cases there is a displacement of iron (step 8 in Fig. 3).
the water molecule present as the sixth ligand and heme X-ray crystallography has largely contributed to the
iron becomes pentacoordinated. Also, it moves out of the understanding of how the protein scaffold and flexibility
heme plane of about 0.3 Å and is in a high spin state, as contribute to the main steps of the catalytic cycle and
demonstrated by spectroscopic characterization (Fig. 3) oxygen activation.
(Poulos et al. 1985). The change in the iron electronic First, it is nowadays well known that a reorganization of
state upon substrate binding is known to shift the reduc- the active site takes place upon substrate binding and it is
tion potential toward more positive values, promoting the generally induced by a conformational change of the pro-
first electron transfer from the redox partner to the Fe(III) tein, mainly involving two helices (F and G) that are
that becomes Fe(II) (step 2 in Fig. 3). In the next step connected by a loop (F–G loop) of variable length and
(step 3), the molecular oxygen binds the high-spin heme flexibility. The movement of this part of the protein is
iron, giving a low-spin hexacoordinated iron state, the responsible for opening and closing the substrate access
ferric peroxide (Fe3?-OO-) (step 3 in Fig. 3). In step 4, channel through a mouth that is located at the cytosol–
another electron enters and a negatively charged ferric membrane interface in those membrane-bound mammalian
peroxo anion (Fe3?-OO2-) forms. The entrance of a first enzymes that process hydrophobic molecules. Thus, the
proton leads to the formation of an unstable hydroperox- bioavailability of these compounds is increased in this
ide intermediate known as ‘‘Compound 0’’ (step 5). The specific cellular region.

Fig. 3 Schematic catalytic

cycle of a typical cytochrome H 2O
P450 showing the different 8 Low spin
1 H2O
reaction intermediates and the
R
uncoupling routes (dashed
arrows). Comments on the Compound II High spin
different intermediates are
reported in the main text 7
e-
2

H2O O2
Compound I
H2O
2e-, 2H+ H2O2 O2

6 3
- H+ O
-
H+

2 - Ferric peroxide
4 e-
Compound 0 5
H+

Peroxo anion

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Second, high occupancy reaction intermediates have allowed to trap and study the dioxygen complex and to
been generated in the crystalline enzymes at cryogenic understand how oxygen is activated to perform the
temperatures (88–100 K) and rapid diffraction data col- hydroxylation reaction (Nagano and Poulos 2005). In these
lection allowed to capture a series of time-lapsed pictures studies, the crystal structure of the dioxygen intermediate
of the reaction intermediates at atomic resolution (Sch- shows the importance of Thr252 that promotes the proto-
lichting et al. 2000). Moreover, site-directed mutagenesis nation of the oxygen atom (step 5 in Fig. 3) with the help
on key residues allowed understanding their role in oxygen of the water molecule present in the groove of helix I.
activation and catalysis. These studies have been per- When Thr252 is mutated into alanine (Fig. 4b), the enzyme
formed on the bacterial cytochrome P450 101 (P450cam), loses its activity.
an enzyme able to hydroxylate camphor (Schlichting et al. Asp251 is also necessary to have an active enzyme,
2000), and they showed how the enzyme is able to activate since the mutant Asp251Asn (Fig. 4c) is inactive and this
molecular oxygen. The crystal structure of the reduced can be explained by the fact that, in the mutant, the
form of cytochrome P450cam, generated by the addition of important water molecule necessary for the formation of
sodium dithionite to the enzyme crystals, was solved at 1.9 Compound I is not present, due to the absence of Asp251
Å resolution (Schlichting et al. 2000) and showed no sig- that promotes the movement of helix I which promotes
nificant changes compared to the oxidized form of the water access into the active site.
enzyme. In summary, two residues, Thr252 and Asp251, have a
The crystal structure of the second intermediate of the concerted role in the active site of the protein. Thr252
catalytic cycle (ferrous dioxygen complex), generated by accepts a H-bond from the hydroperoxy (FeIII–OOH)
introducing an oxygen flow in the solution containing the intermediate promoting the second protonation on the
protein crystals at cryogenic temperatures (80–100 K), distal oxygen atom, leading to the O–O bond cleavage and
showed the movement of two residues, Thr252 and formation of compound I (FeIV=O).
Asp251, which creates space to accommodate two new
water molecules hydrogen bonded to the oxygen and to the
protein backbone (Fig. 4a). In particular, the shift of 5 Human aromatase: a membrane-bound class II
Asp251 allows for a movement in the I helix upon O2 cytochrome P450
binding that allows the insertion of the two water mole-
cules involved in H-bonding responsible for the O–O bond Human aromatase (CYP19A1), also known as estrogen
cleavage. A water molecule is accommodated in the so- synthase, is a class II cytochrome P450 able to catalyze the
called ‘‘groove’’ of helix I. conversion of androgens into estrogens in the last step of
A few years later, a combination of X-ray crystallog- steroid hormone biosynthesis (Thompson and Siiteri 1974).
raphy, site-directed mutagenesis and activity assays In particular, the enzyme converts androstenedione,

A B C
D251
D251 N251

Wat234 Wat122

Wat228 T252 Wat181 A252 T252

Fig. 4 Details of the crystal structure of the P450 prototype Thr252 (D252) and Asp251 (D251) are also shown with their
P450cam, in its wild type (a), Thr252Ala (b) and Asp251Asn respective mutants. The substrate camphor is shown in its bound
(c) mutants. The heme iron is shown in orange, the oxygen of the position above the heme. The effects of the mutations in the structure
catalytically relevant water molecules (Wat122, Wat181, Wat228 and and activity of the enzyme is commented in the text
Wat234) is shown as cyan spheres, the side chains of the key residues

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testosterone and 16a-hydroxytestosterone into estrone, the polypeptide chain are organized in a fold with a char-
estradiol and estriol, respectively. acteristic triangular shape formed by 12a-helices (A to L)
It is a membrane-bound enzyme present in the endo- and 10b-strands (1 to 10), organized into four b-sheets,
plasmic reticulum of most tissues and organs of the human surrounding a type B heme cofactor (Fig. 5a).
body, including ovaries, testis, brain, adipose tissue and The heme cofactor is accommodated in a catalytic pocket
bones. Being the enzyme that converts male sexual hor- of 400 Å3 very tightly compared to other human cyto-
mones into females one, the role of the enzyme is crucial in chromes P450 such as P450 3A4 (530 Å3) and 2D6 (540 Å3).
sex dimorphism, development and reproduction. Moreover, The active site of the enzyme (Fig. 5b) contains the
the levels of the estrogen products of the enzyme are heme cofactor where the iron atom is bound to Cys437 and
known to be altered in some pathologies, such as breast interacts with Arg115, Trp141, Arg375 and Arg435. The
cancer and endometriosis (Santen et al. 2009). Aromatase peculiarity of the active site of the protein is the presence
inhibitors are currently used for breast cancer treatment in of a unique proline residue (Pro308) in the helix I that
post-menopausal women. causes a shift in the helix axis necessary to accommodate
Aromatase is also highly expressed in the brain (Naf- the androgen substrate (Ghosh et al. 2009).
tolin et al. 1975) and is part of the so-called neuroendocrine The 17-keto oxygen of androstenedione forms a hydro-
system. High levels of the enzyme are present within dif- gen bond of 2.8 Å with the backbone amide of Met374 and
ferent areas of the brain, including hippocampus, amygdala the 3-keto oxygen atom of the substrate forms a hydrogen
and several other regions of the cerebral cortex (Naftolin bond 2.6 Å long with Asp309. To do so, this residue was
et al. 1975; Wagner and Morrell 1997). Estrogens produced suggested to be protonated at physiological pH.
by brain aromatase are known to play important roles for In 2013, the crystal structure of recombinant human
axon growth and migration, regulation of neural plasticity aromatase truncated at the N-terminus (rArom) in complex
(Davis et al. 1996; Horvath and Wikler 1999), protection with the substrate androstenedione was analyzed at a res-
against neurodegenerative pathologies like Alzheimer’s olution of 3.29 Å (Lo et al. 2013). The recombinant
and Parkinson’s diseases and stroke (Garcia-Segura et al. enzyme shows a compact structure, superimposable to the
2001; Roselli 2007). Moreover, they influence learning, one of the placental enzyme. However, to accommodate
mood, memory, sexual behavior and reproductive endo- substrates as well as inhibitors such as the ones used for
crine functions by acting as neurotransmitters and neuro- breast cancer treatment, some conformational changes
modulators (Fink et al. 1998; Roepke et al. 2011; must take place at least in those regions involved in ligand
Balthazart and Ball 2006). access. A combination of methodologies aimed at studying
the dynamics and flexibility of proteins in solution was
5.1 Crystal structure of human aromatase applied to human aromatase to check if the flexibility of the
enzyme is affected by ligand binding. In particular, H/D
The first crystal structure of full-length placental human exchange followed by FTIR spectroscopy and time-re-
aromatase in complex with the substrate androstenedione solved fluorescence were used to measure the kinetics of
was published by Ghosh and co-workers in 2009 at a res- H/D exchange when aromatase was in the ligand-free form
olution of 2.9 Å and then refined at the resolution of 2.75 Å and when complexed with a substrate and an inhibitor (Di
in 2012 (Ghosh et al. 2009, 2012). The 503 amino acids of Nardo et al. 2013). Our group demonstrated that the

A B
Asp309
Met374 Pro308
2.6 Å

2.8 Å

Helix I

Fig. 5 a Crystal structure of full-length placental human aromatase loops in grey, surrounding the type B heme cofactor in red. b Details
in complex with the substrate androstenedione showing the twelve a- of the enzyme active site showing the interactions of the substrate
helices in blue ribbon, the ten b-strands in magenta and the disordered androstenedione (blue) with key residues (Met 374 and Asp309)

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flexibility of the protein scaffold is decreased in the pres- crystal structure shows that the C19 is the closest atom to
ence of a ligand. In particular, the substrate androstene- the heme center and the binding pocket is composed of an
dione and the inhibitor anastrozole induce a significant oxidation site that includes heme and Thr310 and a pro-
decrease in the H/D exchange rate constant in a-helices tonation site that includes Asp309. This residue is sug-
suggesting a lower flexibility. gested to be involved in a hydrogen bond with the 3-keto
moiety of the substrate and also to the proton donor for the
5.2 The catalytic mechanism final aromatization step.
Thanks to the development of a robust recombinant
Among cytochromes P450, human aromatase shows one of system of human aromatase and a combination of spec-
the most complicated reaction mechanisms involving mul- troscopic and site-directed mutagenesis studies, our group
tiple steps where the enzyme acts as a hydroxylase and a was able to estimate a pKa of 8.2 for this residue (Di Nardo
lyase. Aromatase has a very narrow substrate specificity that et al. 2015a). Moreover, we demonstrated that the mutant
relies on the architecture of its active site that is comple- Asp309Asn, where the protonable group of aspartic acid
mentary to the androgen substrate and justifies the very high was removed due to the introduction of a non-protonable
affinity for the substrate (nM range) compared to other residue, is inactive indicating a key role in donating the
P450s. Since estrogens are responsible for reproduction and proton to the androgen substrate for the aromatization of
key for life, this enzyme has a very high substrate specificity the A ring of the steroid (Lo et al. 2013).
and a fine catalytic mechanism because the enzyme is One of the questions about enzymes catalyzing multi-
involved in a vitally important metabolic reaction. step reactions is their ability to retain and process the
The overall reaction requires three cycles, each one substrate and the following reaction intermediates in the
requiring one mole of molecular oxygen and one mole of active site and only to release the final product (processive
NADPH as electron donor. After a first oxidation at the reaction), or whether the intermediates, according to their
C19 methyl group, the aromatase catalyzes a second concentration, can freely dissociate and be eventually re-
hydroxylation at C19 which removes the 19-pro-R hydro- accepted to be further processed (distributive reaction).
gen to yield 19-gem-diol. This intermediate spontaneously This point was addressed for aromatase by pulse chase
dehydrates to produce an aldehyde. Then the enzyme acts experiments (Sohl and Guengerich 2010) as well as bio-
as a lyase catalyzing the oxidative cleavage of the C10– electrochemistry (Di Nardo et al. 2015b). In the latter case,
C19 bond to produce an aromatic estrogen compound and the enzyme was immobilized on glassy carbon electrodes,
formic acid (Fig. 6). and electrode-driven catalysis was conducted in the pres-
The mechanism of the final aromatization step is still an ence of excess of substrate and at a non-saturating substrate
object of debate and different models have been proposed concentration. In the first case, the enzyme produced only
and reviewed (Di Nardo and Gilardi 2013). However, the the first intermediate, since it prefers to continue to use this

Fig. 6 Reaction intermediates

resulting from the three
consecutive catalytic cycles
carried out by human aromatase
required for the conversion of
the substrate androstenedione in
the final products estrone and
formic acid

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Fig. 7 Two modes of binding

for aromatase inhibition.
a Irreversible inhibition by

N
exemestane and b quasi- Exemestane Anastrozole
O
irreversible inhibition by CH3

CH3
anastrozole

1
N

2
N
O

N4
CH2

IRREVERSIBLE INHIBITION QUASI-IRREVERSIBLE INHIBITION

compound rather than processing the first intermediate into consequence, much attention must be paid in not altering
the second one. In fact, the affinity for the first substrate is their delicate structure during their technological
higher than that measured for the first intermediate. When exploitation. The wild-type enzymes have already been the
the starting substrate concentration is lowered, the first subject of applications in the biosensing, bioanalytical and
intermediate formed can compete with it and therefore the pharmaceutical areas (Sadeghi et al. 2011; Fantuzzi et al.
second intermediate and the final products are detected. 2004, 2011; Panicco et al. 2011).
These data showed that the intermediates of aromatase Their function can also be modulated by mutagenesis,
reactions freely dissociate according to the concentrations changing specific amino acids by site-directed mutagenesis
and therefore the aromatase reaction follows a distributive and/or directed evolution. This allows expansion of their
mechanism (Di Nardo et al. 2015b). application in the widest industrial fields.
Another important topic of research in the aromatase
field is the development of inhibitors for breast cancer
treatment. In particular, these drugs are designed to block References
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