Tài liệu Báo cáo khoa học: Models and mechanisms of O-O bond activation by cytochrome P450 A critical assessment of the potential role of multiple active intermediates in oxidative catalysis doc

REVIEW ARTICLE
Models and mechanisms of O-O bond activation by cytochrome P450
A critical assessment of the potential role of multiple active intermediates
in oxidative catalysis
Peter Hlavica
Walther-Straub-Institut fu
¨
r Pharmakologie und Toxikologie der LMU, Mu
¨
nchen, Germany
Cytochrome P450 enzymes promote a number of oxidative
biotransformations including the hydroxylation of unacti-
vated h ydrocarbons. Whereas the l ong-standing consen sus
view of the P450 mechanism i mplicates a high-valent iron-
oxene species as the p redominant oxidant in the radicalar
hydrogen abstraction/oxygen rebound pathway, more
recent studies on isotope partitioning, product rearrange-
ments with Ôradical clocksÕ, and the impact of threonine
mutagenesis in P450s on hydroxylation rates support the
notion of the nucleophilic and/or electrophilic (hydro)
peroxo-iron intermediate(s) to be operative in P450 catalys is
in addition to the electrophilic oxenoid-iron entity; this may
contribute to the remarkable versatility o f P 450s i n sub strate
modification. P recedent to t his mechanistic conc ept is given
by studies with natural and synthetic P450 biomimics. While
the concept of an alternative electrophilic oxidant necessi-
tates C-H hydroxylation to b e brought about by a cationic
insertion p rocess, recent calculations employing density
functional theory favour a Ôtwo-state reactivityÕ scenario,
implicating the usual ferryl-dependent oxygen rebound
pathway to proceed via two spin states (doublet and quar-

tet); s tate crossing is thought to be associated with either an
insertion or a radicalar mechanism. Hence, challenge to
future strategies should be to fold the disparate and some-
times c ontradictory d ata i nto a harmonized overall picture.
Keywords: ( hydro)peroxo-iron; iron-oxene; O
2
-activation;
P450 biomimics; P450.
Introduction
Cytochrome P450 (P450 or CYP) enzymes (EC 1.14.14.1),
a superfamily of b-type hemoproteins found in organisms
from all domains of life [1], a re major catalysts in the
oxidative biotransformation of a structural diversity of
endogenous and exogenous compounds [2]. While the
general chemistry of substrate hydroxylation has been
assessed on a broad basis, the specific problem of dioxygen
activation during P450 cycling is still the most important
and intriguing one in the area of P450 research. Here, the
need for an active oxidant capable of insertion into
unactivated C-H bonds in hydrocarbons and related
compounds has extensively captured the i magination of
biochemists owing to the unfavourable th ermodynamics of
the dissociation e vent [3]. Early views of such a m echanism
focused on an oxygen insertion pathway promoted by an
electrophilic, h igh-valent iron-oxo spe cies (compound I) [4].
This hypoth esis was soon supplanted by the Ôhydrogen
abstraction/oxygen rebound Õ concept implicating the exist-
ence of radical intermediates, a s d eveloped on the basis of
the well-known chemical p roperties of peroxidases and
porphyrin m odel systems [5,6]. The m echanistic details of

oxygen transfer have been addressed elsewhere [7,8].
Mounting evidence provided during t he past decade
suggests that hydroxylation reactions are more c omplex
than previously anticipated, and are not compatible with
the idea of a single reaction pathway. The picture began to
cloud when the application o f ultrafast Ôradical clocksÕ
to time the oxygen-rebound step disclosed the amounts o f
rearranged products not to correlate with the radical
rearrangement rate constants [9]. Moreover, the use of a
probe that could d istinguish between radical and cationic
species hinted at the interference of cationic r earrangements,
predicting the hydroxylation to occur via an insertion
reaction in place o f abstraction and recombination [9]. The
former process thus necessitated the insertion into a C-H
bond of the elements of OH
+
, implying that the ultimate
electrophilic oxidant was either hydroperoxo-iron or iron-
complexed hydrogen p eroxide [10]. In addition, examina-
tion of the o xidative deformylation o f cyclic aldehydes as a
model for the demethylation reaction mediated by steroido-
genic P450s strongly favoured nucleophilic attack on the
Correspondence to P. Hlavica, Walther-Straub-Institut fu
¨
r Pharmak-
ologie und To xikologie, G oethestr. 33, D-80336 Mu
¨
nchen, Germany.
Fax: +49 8 9 218075701 , Tel.: +49 89 218075706,
E-mail:

Abbreviations: TSR, two-state reactivity; KIE, kinetic isotope effects;
Hb, haemoglobin; Mb, myoglobin; HO, heme oxygena se; PDO,
phthalate dioxygenase; TDO, toluene dioxygenase; NDO, naphtha-
lene 1,2-dioxygenase; PMO, putidamonooxin; BLM, bleomycin;
NOS, nitric oxide synthases.
Enzymes: Cytochrome P450 (EC 1.14.14 .1); NADPH-cytochrome
P450 oxidor eductase (EC 1.6.2.4); he me oxygenase (EC 1. 14.99.3);
phthalate o xygenase reductase (EC 1.18.1); phthalate dioxygenase
(EC 1.14.12.7); t oluene dioxygenase ( EC 1.14.12.11); naphthalene
1,2-dioxygenase (EC 1.14 .12.12); putidamonooxin (EC 1.14 .99.15);
nitric oxide synthases (EC 1.14.13.39).
(Received 2 9 July 2004, revised 27 September 2004,
accepted 28 September 2004)
Eur. J. Biochem. 271, 4335–4360 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04380.x
substrates by an iron-peroxo intermediate [11]. The sum of
these findings points at the involvement of more than one
active oxidant in the diverse types of P450-catalyzed
substrate processing [12–15].
The g oal of t he p resent perspective is to p rovide a critical
update of several aspects of the c urrent state o f biochemistry
relating to the apparently complex machinery of dioxygen
activation, which is considered to possibly implicate m ul-
tiple oxygenating species in P450 catalysis. Emphasis will be
put on the evaluation of comparative s tudies with non-P450
hemoproteins, nonheme metalloenzymes as well as bio-
mimetic model systems to dis cuss the Ômultiple oxidantÕ vs.
the Ôtwo-state reactivityÕ theory.
Iron-oxene acting as an electrophilic oxidant
in P450-catalyzed hydroxylations
The consensus m echanism for hydrocarbon hydroxylation

by P450 enzymes involves hydrogen atom abstraction from
the hydrocarbon by a high-valent iron-oxo species, best
described as an O ¼ Fe(IV) porphyrin p-cat ion radical,
followed by homolytic substitution of the alkyl radical thus
formed in the so-called Ôoxygen reboundÕ step [5–8]
(Scheme 1). Using C YP2B isoforms as the catalysts, r adical
collaps was demonstrated to occur at highly variable rates
exceeding those of the gross molecular motions of many
enzyme-bound substrates and d epending on the stereo-
chemical specificities of the com pounds to be acted upon
[16,17]. Reduction of ferric P450 to the ferrous state s ets the
stage for d ioxygen binding, the event that commits the
hemoprotein to the step-by-step production of the active
oxidant (Scheme 2). Association of dioxygen with ferr ous
microsomal CYP1A2 [18], certain CY P2B isoforms [19–21],
and CYP2C3 [18] to yield hexacoordinate low-spin com-
plexes has been shown to be characterized by absorption
bands around 420 and 557 nm in the absolute spectra and
broad maxima at about 440 and 5 90 nm in the difference
spectra. Similar optical perturbations were also observed
upon O
2
binding to so-called c lass I P450s, comprising
mitochondrial and bacterial isozymes such as CYP11A1
[22–24] and C YP101 [25,26], r espectively. The rapid initial
step in molecular oxygen activation by both class I and class
II P450s, as measured at varying temperatures, usually
exhibits monophasic kinetic behaviour, with the second-
order rate constants ranging from 0.58 to 8.41 · 10
6

M
)1
Æs
)1
[18,20,24,25]. Interestingly, the presence of certain substrates
such as aromatic amines appears to favour homotropic
cooperativity in dioxygen b inding to P450s: using liver
microsomal samples f rom untreated rab bits, the O
2
satura-
tion kinetics for acetanilide 4-hydroxylation have b een
reported to b ear sigmo idal character c orresponding to a Hill
interaction coefficient, n, of 2.2 [27]. Similar experiments
with N-alkyl arylamines gave concave upward double-
reciprocal plots of v elocity vs. O
2
concentration, from which
n could be calculated to have a value of 2.0–2.1 [28,29].
Apparent cooperativity in dioxygen association was found
to be highly sensitive to changes in hydrogen io n concen-
tration a nd was most pronounced at physiological pH,
whereas CO, acting as a positive effector, abolished
autoactivation at all pH values examined (Fig. 1) [ 30]. In
view of the well-known microheterogeneity of s everal rabbit
liver P450s [31], t he amine-induced co operativity in O
2
complexation has been argued to involve the equilibrium
between multiple , kinetically distinct protein conformations
[32]. A lternatively, the oligomeric nature of P450 [33] might
offer the possibility of substrate–specific subunit i nter-

actions, a s has been proposed for the fractional saturation
of hemoglobin by dioxygen [34].
Results from resonance Raman spectroscopy [35] and
Mo
¨
ssbauer studies [36] with microbial CYP101 indicate that
Scheme 1. Rebound mechanism for P450-catalyzed hydroxylations.
Reproduced from [6] with permission.
Fe
(III)
Fe
(II)
Fe
(III)
O
O
Fe
(III)
O
O
Fe
(III)
O
O
H
Fe
(IV)
O
Fe
(III)

Fe
(III)
HO
OH
Fe
(IV)
O
HO
OH
+ H+
+ H+
+ H+ H
2
O
O
2
peroxo-iron
nucleophilic oxidant
hydroperoxy-iron
(inserts OH
+
?)
oxo-ion, low spin
(inserts O)
spin inversion
iron-complexed hydrogen peroxide
(inserts OH
+
?)
oxo-ion, high spin

(abstracts H
+
)
e
e
Scheme 2. The putative i ron-oxygen inter-
mediates in P450 a nd their possible roles as
oxidants. D ata collated from [10,15] with
permission.
4336 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
the ÔoxyÕ intermediate o f P450 most likely e xists in the low-
spin ferric-superoxide form, with the sixth 3d electron
largely transferred to O
2
in an autoxidative process
(Scheme 2). Spontaneous autodecomposition of oxy-cyto-
chrome 2B4 to r elease ferr ic pigment and superoxide [37]
has been shown to o ccur in a b iphasic [21,38] or even
triphasic [39] fashion, while m onophasic fi rst-order kinetics
were observed for autoxidation of substrate-bound adr eno-
cortical CYP11A1 [23,24] and bacterial CYPs 101 an d 102
[25,26,40], as measured above 0 °C or at s ubzero temper-
atures. Abortive decay of oxygenated P450 is r etarded in the
presence of hydroxylatable substrate [23,26,38], preserving
the complex for arrival of the second electron, and is
inversely proportional to the coupling efficiency of the
system [41]. M oreover, the steady-state level of oxyferrous
P450 has been recognized to be governed by the hydrogen
ion concentration a nd ionic s trength of t he reaction medium
[21,24,25]. In view of the strategic importance of the

oxyferro intermediate in the p rocess of dioxygen ac tivation,
the influence o f the physiological redox partner, cytochrome
b
5
, on its autoxidative breakdown has been examined in
detail: though i ncreasing the rate of regeneration of ferric
enzyme from oxygenated CYP2B4 by a factor o f about 8 ,
reduced donor protein added to the assay mixtures failed t o
undergo substantial reoxidation, suggesting the electron
carrier t o act as an allosteric eff ector in this reaction [38,42].
In accord with this, both apocytochrome b
5
and aporubre-
doxin reportedly stimulate autoxidative transformation of
oxy CYP101 to the ferric state [43]. Superoxide departing
from regenerated P450 h as been fo und to serve a s a source
of hydrogen peroxide usually generated during NADPH/O
2
consumption [44].
Addition of an electron to oxyferrous P450 (Scheme 2)
results in the formation of an optimized species,
37 kcalÆmol
)1
higher in energy, with elongated Fe-O
distance but unchanged O-O bond characteristics [45].
Significant O-O bon d weakening occurs upon protonation,
the calculated proton affinity being )442.1 kcalÆmol
)1
[45].
The proton-delivering machinery has been recognized to

involve a highly con served active-site threonine residue
[46,47] working in tandem with an essential a spartate [48–
50]. The residue pair has been ascribed a critical role in
orchestrating the dynamic organization of a ctive-site water
molecules [46], f orming a h ydrogen-bonded network
capable of pumping protons to the reduced FeO unit [51]
to generate the h ydroperoxo-iron d erivative ( compound O;
Scheme 2). Intermediacy of the end-on Fe(III)-OOH species
has been unequivocally proven by electronic absorption,
EPR and ENDOR spectroscopic techniques upon cryo-
radiolytic reduction of oxy CYP101 [52–55] and CYP119
[56] at 77 K. The same intermediate was also obtained by
reacting ferrous CYP101 with KO
2
[57] or bioreduction of
oxyferrous CYP101 with putidaredoxin [58].
Unless the protonated peroxide complex decays in a
nonproductive mode to liberate ferric enzyme and H
2
O
2
[18], conversion to the actual oxidant proceeds with a
significant energy release of 50 kcalÆmol
)1
[59]. While
acylation of the distal oxygen to m ake i t a better leaving
group prior to Fenton-type homolytic O-O bond rupture
has been vitiated owing to discrepancies b etween theory and
measured data [60], t he most favoured a ctivation pathway is
heterolytic O-O bond scission to formally produce a

[FeO]
3+
species (Scheme 2) [6,8], having a midpoint poten-
tial of 1.5–2.0 V [61]. The so-called ÔpushÕ effect of the
thiolate ligand in P450s has been shown to promote
heterolytic cleavage of heme-bonded dioxygen b y increasing
electron density at the iron atom [62–66]. The electron-
donating properties of the active-site thiolate of CYP101
have been demonstrated to be enhanced by putidaredoxin-
induced alterations in enzyme conformation [50–67].
Attempts were mad e to characterize the P450 reactive
oxygen intermediate. Thus, i odosylbenzene, a single-oxygen
donor [68], as well as peroxides and peracids, acting as
versatile O
2
surrogates in oxidative reactions [69–71], have
been revealed to elecit spectral perturbations with P450s
closely resembling those of the green, high-valent FeOPor
•
+
species (compound I) of peroxidases, including the thiolate-
ligated, P450-like chloroperoxidase enzyme [72–75]. These
findings lent credit to the notion, that an analogous key
oxidant might be operative in P450-catalyzed monooxy-
genations, too, a lbeit t here is a significant difference between
P450 and peroxidase models regarding the displacement of
the iron atom from t he porphyrin plane, resulting in longer
Fe-O bond in the P450 active intermediate [76]. Density
functional studies demonstrate that both enzyme systems,
though looking very similar, behave like chemical chame-

leons, in which small alterations in the environment can
cause drastic changes in the reactivity of the active species
[76]. Further support in favour of th e idea of the involve-
ment of a high-valent iron-oxene in P450 catalysis came
from experiments with metalloporphyrin models [5,6,77]. Of
particular importance, a green oxo-ferrylporphyrin p-cation
radical intermediate could be isolated and spectrophoto-
metrically and chemically characterized, that was capable of
Fig. 1. Effe ct of hyd rogen i on concen tration o n the Hill inte ractio n
coefficient n for oxygen b inding. Rabbit liver microsomal N-oxid e for-
mation from N,N-dimethylaniline was measured in the a bsence (d)and
presence ( s)of490l
M
CO. Reproduced from [30] with permi ssion.
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4337
oxygen transfer reactions [78]. Nevertheless, identification
of the [FeO]
3+
adduct by UV-visible spectroscopic a nalysis
of CYP119 [79] or transient X-ray crystallography using
CYP101 [80] appears to be quite tentative.
The proportion of the putative iron-oxene species not
used for monooxygenations undergoes uncoupling to
generate ferric P450 and wat er [ 81] in a 4-electron r eductiv e
process [82], uncoupling being controlled by substrate
accessibility [83]. In fact, the presence of substrate has been
shown to s tabilize the active oxy c omplex pro duced with
CYP2B4 and organic hydroperoxide, and the protective
effect is intensified by cytochrome b
5

binding [84,85]. Active
oxidant thus preserved is thought to promote hydrogen
transfer from substrate t o i nitiate monooxygenation
(Scheme 1 ); this step, which proceeds with a remarkable
low free–energy barrier, has been suggested t o be governed
by peripheral heme substituents in the P450 molecule [86].
Firm evidence for the nonco ncerted hydrogen abstraction/
oxygen rebound chemistry presented in Scheme 1 is provi-
ded by a plethora of experimental observations such as (a)
the stereochemical scrambling in norbornane [87] and
camphor [88] hydroxylation ( b) the allylic rearrangements
found in the hydroxylation of unsaturated hydrocarbons
[89] (c) the correlation of susceptibility toward oxidative
attack with C-H bond strength [90] (d) the large kinetic
isotope effects (KIE; k
H
/k
D
% 11) for C-H a ctivation u sing
norbornane [87], diphenylpropane [91] or difluorocamphor
[92] as the hydroxylatable substrates and (e) the results from
investigations with Ôradical clockÕ probes such as bicyclo-
pentane, having highly strained carbocyclic structures to
permit the unmasking of radical intermediates that rear-
range at a rate faster than that of the r ecombination step
[16,93].
Despite the apparent predominance of the h ydrogen
transfer mechanism as t he initial s tep in s ubstrate hydroxy-
lation, electron transfer to g enerate a carbocation, followed
by capture of a hydroxyl anion h as been discussed as an

alternative oxygenating principle [94,95]. The net outcome
would be oxidation of an otherwise unactivated C-C s ingle
bond. Although cations may be the logic precursor for
certain substrates with low oxidation potentials, such a
pathway cannot b e reconciled with the large K IE and
stereochemical scrambling detaile d above. To quantitative ly
assess the significance of electron transfer in the transition
states of hydroxylation r eactions, studies on the regioselec-
tivity of nitroacenaphthene oxygenation were conducted
with various oxometalloporphyrins; hydrogen abstract-
ion was shown to be the preferred route for all models
examined [96].
Hydroperoxo-iron acting as an alternative
electrophilic oxidant in P450-catalyzed
hydroxylations
Evidence from kinetic analysis of P450 function
Studies on the oxidative transformation of 1-methyl-2-
phenylcyclopropane and its mono-, di-, and trideuterio-
methyl congeners by microsomal CYP2B1 and CYP2E1
suggested that, judging f rom the large magnitudes o f the
combined primary and secondary KIEs for hydrogen
abstraction, rotation in the enzyme pocket was faster than
its relatively slow reaction (< 10
6
Æs
)1
) with the putative
iron-oxene species [97], while the lifetimes o f carbon-centred
radicals derived from a diverse set of substrates are on the
order of about 10

)10
s [98]. Moreover, the randomness of
the apparent intramolecular KIEs for unrearranged and
rearranged alcoh ol products generated from e nantiomeric
dideuteriomethyl substrate forms implicated that more than
one reaction channel existed [99]. This concept was
reinforced when the KIEs for NADPH-and cumene
hydroperoxide-driven N -demethylation of amitryptiline by
CYP2D6 were found to be severely discrepant [100].
Examination of the competitive i ntermolecular KIE fo r
sulfoxidation/N-dealkylation reactions mediated b y bacter-
ial C YP102 hinted at t he involvement of two distinct
electrophilic oxidizing species [101], as was also concluded
from the intermolecular noncompetitive KIEs for a-and
b-hydroxylation of fatty acids by CYP152 peroxygenase
isozymes [102].
Probing of the metabolism of norcarane by CYP2B4
revealed the formation of a cation-derived rearrangement
product not compatible with the hydrogen abstraction
mechanism [103]. The latter was also challenged by the
finding that evaluation of the m etabolic transformation of a
series of cyclopropane derivatives by CYP2B4 gave unrea-
sonably high rate constants for oxygen rebound ( k
OH
)
ranging from 1.5 to 7 · 10
12
s
)1
; this disparate result was

rationalized by possible steric effects in the enzyme’s active
site causing overestimation of the k
OH
values [17]. However,
experiments on the CYP2B1-catalyzed hydroxylation of a
new constrained substrate, that would be less likely to be
subject to steric constraint, also yielded an incredibly high
apparent k
OH
value of 1.4 · 10
13
s
)1
[104]. Moreover, the
plot of the ratio of rearranged to unrearranged alcohol
products vs. the rate constant for rearrangement of the
putative radical intermediate (k
r
) revealed a lack of corre-
lation between these p arameters [104]. In addition, hyper-
sensitive radical probe studies with four P450 isozymes gave
consistently small amounts of rearranged products, ham-
pered radical ring openin g on steric grounds being unlikely
[105]. The sum of these findings thus suggested that there
was either a n error in the kinetic scale f or fast radical
reactions or the mechanistic paradigm of P450-mediated
hydroxylations was i ncomplete. To solve this problem,
further hypersensitive r adical probe substrates were intro-
duced, that could distinguish between radical and carbo-
cation intermediates o n the basis of the identity of the

rearranged products [9,106,107]. Oxidation of these probes
with several members of the CYP2 f amily gave cation-
derived rearrangement products, disproving t he assumption
that such rearrangements arose e xclusively from rad ical
species. Variable partitioning between the radical and
carbocation m echanisms thus was concluded to explain
thewiderangeofk
OH
values described above [106]. From
the small amounts of radical rearrangement products
generated from the hypersensitive probes, the radical
lifetimes in the P450-catalyzed reactions could b e calculated
to range f rom 70 to 2 00 fs [106,107], which a re too short f or
true radical i ntermediates, but rather correspond to vibra-
tional lifetimes or the lifetimes of transition states. Hence,
the c ationic intermediates observed could be ruled out to
originate from oxidation of such transient radicals, so that
their o ccurrence necessitated another mechanistic enigma.
4338 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
In this regard, the most plausible premise is insertion of
OH
+
into a C-H bond to generate protonated a lcohol
species that can undergo solvolysis-type reactions to yield
cationic rearrangement products [9,107]. This route requires
heterolytic O-O bond fission of the hydroperoxo-iron state
of P450 (Scheme 3 A) to release OH
+
and [FeO]
+

[106,107].
However, density functional a nalysis o f mechanisms
involved in ethylene epoxidation by a Fe(III)–OOH model
disclosed barriers for the various pa thways of 3 7–53 kcalÆ
mol
)1
[108]. This was taken to indicate that hydroperoxo-
iron, as such, could not be the u ltimate oxidant, i n line with
its significant b asicity and poor electron-accepting capabil-
ities [108]. Moreover, molecular o rbital calculations c arried
out with a similar model system unveiled nonrep ulsive
potential curves only for peroxo-iron, but not for hydro-
peroxo-iron as the catalytic intermediate in the turnover of
aniline and fluorobenzene [109]. Comparative investigations
on the NADPH/O
2
- and iodosylbenzene-dependent meta-
bolism of lauric acid by C YP2B4 favoured the F e(III)-H
2
O
2
complex (Scheme 3B) as acting as an alternative electro-
philic oxidant [110]. This postulate is in accord with data
from measurements with hypersensitive radical clocks
[9,106], albeit there is some objection to this i dea: protona-
tion of the proximal oxygen in the reduced ferrous dioxygen
unit is usually thought to trigger Fe-O bond weaking
followed by uncoupling of monooxygenation reactions
[111]. On the other hand, stable end-on iron(III )-hydrogen
peroxide complexes have been shown to incur in the

catalytic cycle of cytochrome c peroxidase [112], horseradish
peroxidase [113] and chloroperoxidase [114], but their
immediate participation in monooxygenation processes
has not been established. Finally, molecular dynamics
simulations employing the CYP101 crystal structure pro-
posed the d iprotonated species displayed in Scheme 3C to
be an oxidant far superior to compound I [115]. As can be
readily seen, the question of the nature of the alternative
oxygenating intermediate remains inherently elusive.
The functional importance of hydroperoxo-iron or iron-
coordinated hydrogen peroxide as the putative second
oxidant i n P 450 catalysis is also c orroborated b y s tudies on
heteroatom oxidation. Thus, comparative investigations
on the NADPH/O
2
- and cumene hydroperoxide-driven
N-hydroxylation of 4-chloroaniline by CYP2B4 indicated
discrepancies in the positions of the Soret maxima in the
absolute spectra o f the individual oxy complexes [ 116].
Noteworthy, transformation o f P450 to t he denatured P420
form through t reatment with either p-chloromercuribenzo-
ate or d eoxycholate rendered t he hemoprotein a more
powerful peroxygenase [116], but disrupted NADPH-linked
monooxygenase activity [117]. Hence, resonanace stabiliza-
tion via the thiolate Ôpush e ffectÕ (see above) did not appear
to be obligatory when peroxide s ubstituted for reduced
cofactor and d ioxygen. While N-(4-chlorophenyl) hydroxy-
lamine was found to be the major metabolic product under
mixed-function conditions, a marked change to the prepon-
derant formation of 1-chloro-4-nitrobenzene w as observed

when organic hydroperoxide served as the oxygen donor
[116]. Involve ment i n the N-oxidative p rocess o f CmO
•
(CmO
Æ
2
) radicals could be safely ruled out owing to
insensitivity of the reaction toward radical scavengers,
whereas blockage of turnove r by cyanid e hinted a t an
iron-based m echanism [116]. The sum of these findings
raised serious questions as to the commonness of the
oxygenating s pecies operative in the NADPH- and hydro-
peroxide-sustained hydroxylations. In fact, evidence has
been provided for the existence of fairly s table Fe(III)-OOR
intermediates generated by reacting organic hydroperoxides
with mononuclear iron catalysts [118–120] or intact
CYP2C11 [121], a nd their ability t o transfer oxygen to
substrates prior to h eterolytic cleavage at low t emperatures
has been ascertained [122–124]. A s N-hydroxylation of
4-chloroaniline by the putative Fe(III)-OOR species must
compete not only with conversion of the intermediate to
[FeO]
3+
, but also with self-destructive oxidation of the heme
moiety of P450 [125], it seems w orth mentioning that the
rate of cumene hydroperoxide-induced loss of CO-reactive
CYP2B4 [85] could be demonstrated to be far below that of
release of N-oxy product from the ternary c omplex [116].
There is also reason t o e nvisage iron -bound hydro-
peroxide as a potential oxidant in NADPH-promoted

N-oxygenation of N,N-dimethylaniline by C YP2B4: the
presence of superoxide dismutase inhibits the r eaction by
75%, whereas catalase o r mannitol l eave N-oxide f ormation
unaffected, dismissing fre e H
2
O
2
or OH
•
radicals to act as
catalysts [126]. Notably, investigations with a superoxide-
generating system ruled out O
2
•
)
itself to function as the
active intermediate, so t hat superoxide was invoked to s erve
as a source f or production of the ultimate o xygenating
species, presumably Fe(III)-OOH, catalyzing attack on the
electron-rich nitrogen centre of the tertiary arylamine
[126–128]. T he active oxidant thus was anticipated to arise
from interaction, in the p resence of protons, of newly
generated O
2
•
)
with either ferrous or oxyferrous [Fe(III)-
O
2
•

)
] P450, as given in Eqns 1 and 2 [129–132]. That Fe(III)-
OOH generated in this way w ould only serve as a precursor
in the transformation to:
FeðIIÞþO
ÆÀ
2
þ H
þ
!½FeðIIIÞÀOOH
À
ð1Þ
½FeðIIIÞÀO
ÆÀ
2
þO
ÆÀ
2
þ H
þ
!½FeðIIIÞÀOOH
À
þO
2
ð2Þ
iron-oxene as the actual catalyst could be discounted
on kinetic grounds. As an example, the reaction
sequence given in Eqn 2 follows second-order kinetics
with a rate constant of 4 · 10
3

M
)1
Æs
)1
[133], while
injection into Fe(II)-O
2
of the ÔsecondÕ electron to
produce compound I during regular catalytic cycling is
a diffusion-controlled process characterized by a rate
constant of 4 · 10
10
M
)1
Æs
)1
[134]. Comparison of these
data no doubt precludes the major portion of ferryl
material required for efficient substrate turnover to
originate from the dismutation-type bypass reaction. As
Fe
3
+
O
SCys
OH
Fe
3
+
O

SCys
O
H
Fe
3
+
O
SCys
OH
H
Fe
3
+
O
SCys
OH
H
H
A
B
C
Scheme 3. Potential Ôsecond oxidantÕ species in P450 catalysis. Data
adapted f rom [108] w ith permission.
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4339
conversion of the hydroperoxo entity to [FeO]
3+
is a
second-order event, encompassing interaction of the
peroxo intermediate with a proton source to initiate O-O
bond cleavage with water release, the half-life of this

step is inversely correlated with the initial concentration
of Fe(III)-OOH. Hence, the much lower rate of
production of the latter in the superoxide-supported
pathway necessarily results in a depressed level, within
the time scale of the measurements, of hydroperoxo-iron
component relative to the standard redox situation
(Scheme 2). This is likely to cause an increase in both the
half-life of the scission process and the lifetime of
Fe(III)-OOH, possibly fostering direct oxygen insertion
into substrate.
Evidence from experiments with genetically engineered
P450 enzymes
Inferential evidence of two electrophilic oxidants acting as
catalysts in substrate h ydroxylations came from studies with
mutated P 450s. T he crystal s tructures o f bacterial CYPs 101,
102 and 108 contain a highly conserved active-site threonine
within H-bond ing distance t o the per oxo-iron unit [135]. Of
particular interest, attenuated camphor and laurate hydroxy-
lation was observed, when T252/268 in the CYP101 and
CYP102 po lypeptide, re spe ctively, w ere r eplaced with alan-
ine [136,137]. N evertheless, the T252A variant was found to
accept electrons from NADH and reduce dioxygen to H
2
O
2
[137] via the intermediacy o f hydroperoxo-iron [53]. Muta-
tion was considered to disrupt a key step in H
+
delivery,
presumably introduction of the s econd proton to hamper O-

O bond dissociation [53]. T herefore, P450 mutants devoid
of the active-site threonine were regarded ideal m eans for
testing the direct involvement of hydroperoxo-iron in
epoxidations. Indeed, a drastic i ncrease in the ratio of
epoxide to hydroxy products derived from various camphor
analogues during catalysis by the T252A congener of
CYP101 could be demonstrated in comparison to the wild-
type parent [138]. Similar findings were made with truncated
CYPs 2B4 and 2E1 lacking the active-site threonine: the
mutants mediated a lkene metabolism at a n increased ratio o f
epoxidation t o allylic hydroxylation [139].
Using the same wild-type and engineered P450 pairs, the
potential involvement of Fe(III)-OOH in hydroxylation
reactions was inferred from mutant-induced changes in
regioselectivity during the oxidation o f probes designed to
give different r earrangement products with radical and
cationic intermediates [99,105,107,140]. Moreover, t run-
cated CYP2E1 with T303 replaced by alanine was shown to
exhibit considerably higher activity than the parent enzyme
in eliminating p-substituents in phenols to yield hydro-
quinones [141].
The participation of an alternative electrophilic interme-
diate in heteroatom oxygenation was assessed by employing
the T268A mutan t of CYP 102: the engineered enzyme
fostered sulfoxidation of p-(N,N-dimethylamino)thioanisole
relative to N-dealkylation of th e substituted amine function
[101]. A mutant of truncated CYP2B4 with exchange of
alanine for threonine at position 302 turned out to have
decreased ability to catalyze NADPH-dependent N-oxide
formation from N,N-dimethylaniline, questioning an

obligatory h ydroperoxo-iron-promoted mechanism [142].
However, when the m easurements were conducted w ith
iodosylbenzene i n place of NADPH /O
2
to directly generate
the favoured [FeO]
3+
entity [68], the enzyme variant still
mediated N-oxygenation of the tertiary arylamine at a rate
less than half that of the wild-type-catalyzed reaction [142],
so that reasonable interpretation o f the data seems d ifficult.
Evidence from comparative studies with non-P450
hemoproteins and metalloporphyrin models
Hemoglobin (Hb) and myoglobin (Mb). When operative
in its n atural e nvironment, the erythrocyte Hb exerts P 450-
like monooxygenase activity [143] in that the concurrence of
multiple reductase systems permits NAD(P)H-supported
electron transfer to t he pigment [144,145]. In fact, iso-
lated Hb r econstituted with N ADPH-cytochrome P450
oxidoreductase (EC 1.6.2.4) has been demonstrated to bring
about NADPH/O
2
-promoted alkane hydroxylation [146] as
well as N- and O -dealkylation reactions [147], albeit at
considerably lower c atalytic potency as compared with P450
enzymes. Of p articular interest, H b has been found to
mediate formation of p-aminophenol from aniline both in
intact erythrocytes, supplemented with glucose to allow
NADPH production via the pentose phosphate pathway
[148], and in a reconstituted system containing P450

reductase, NADPH and atmospheric oxygen [149]. In the
latter case, c atalase inhibited e nzyme activity by a bout 94%
in the absence or presence of reductase, suggestive of
a hypothetical mechanism for p-hydrox ylation of the
aromatic amine involving H
2
O
2
, formed t hrough
dismutation of autoxidatively generated superoxide, to
produce t he active intermediate, Hb(III)-OOH [149]. I n
line with this, alkaline hemin (ferriprotoporphyrin I X) has
been shown t o activate O
2
to the hydroperoxide anion in the
presence of NAD(P)H [150] with the immediate i nsertion
of oxygen into the benzene ring of aniline to yield
p-aminophenol [151]. Formation o f the oxygenating
species has been recognized to be fac ilitated by the b inding
to HbO
2
of aniline and some of its derivatives, causing
distortion of the iron-oxygen bond to s uch an extent as to
accelerate autoxidation b y alleviating electron transfer from
ferrous iron to O
2
[147,152]. Superoxide displaced from
HbO
2
has been postulated to contribute to production of

the h ydroperoxo-methemoglobin entity by reducing heme-
bound oxygen in the p resence of a proton source as given in
Eqn 2 [131,153]. Indeed, radiolytically reduced samples of
oxygenated Hb [154,155] and Mb [156–158] at cryogenic
temperatures have been shown by EPR studies to generate
the peroxo-bound hemoproteins, with the Fe-O-O unit
being s tabilized by bonding to the distal histidine proton; the
latter was detected to be transferred upon annealing t o give
the hydroperoxo derivatives. Similar results were obtained,
when metmyoglobin was reacted with H
2
O
2
at 77 K [159].
The ability of Hb to catalyze heteroatom o xygenation is
well established. Thus, methemoglobin and H
2
O
2
transform
thianthrene 5-oxide to both the 5,5-and 5,10-dioxide
metabolites [160]. T he finding that most, if not all, of the
sulfoxide o xygen i n the 5,5-dioxide product originates from
hydrogen peroxide but not from
18
O
2
has b een rationalized
by the possible participation in this reaction of a peroxo-
iron catalyst. T his view i s c ompatible with t he capability of

mononuclear peroxo intermediates derived from iron and
4340 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
titanium porphyrin complexes upon treatment with super-
oxide and H
2
O
2
, respectively, to directly promote sulfoxi-
dation reactions [161].
Similarly, Hb has been reported t o perform N-hydroxy-
lation of 4-chloroaniline both in erythrocyte suspensions
[162] and in aerobic systems reconstituted with either
NADPH-P450 reductase or the N ADH-cytochrome b
5
reductase/cytochrome b
5
segment of the electron transfer
chain in the presence of NAD(P)H [ 163,164]. Under t hese
conditions, a ddition to the assays of s uperoxide dismutase or
catalase disrupted N-oxygenating activity by about 70%,
again posing emphasis o n the pivotal role o f H
2
O
2
in forming
the active oxidant. It should b e noted that N-oxidative
metabolism o f 4-chloroaniline is associated with optical
changes characterized by a Soret band at 418 nm in the
absolute spectrum [163], closely r esembling the spectral
perturbations arising f rom reduction of MbO

2
by hydrated
electrons [134] or r eaction of ferrous CYP101 with super-
oxide [57] to y ield Fe(III)-OO(H). Importantly, N-(4-chlo-
rophenyl)hydroxylamine, generated as the primary
metabolic product, has been found to be prone to hydro-
peroxo-methemoglobin-promoted conversion to the 4-chlo-
rophenyl nitroxyl radical [165]. The same mechanism
appears to a lso apply t o one-electron o xidation of the
nitrogen centres in N,N-disubstituted p-phenylenediamines
to give Wurster’s blue aminyl radicals: the HbO
2
-dependent
processes have been shown to b e decelerated by up to 50%
in the presence of catalase, whereas superoxide anion was
likely to be of m inor importance i n formation of the radical
cations [166].
Collectively, the drastic disruption of the C- and
N-oxidative biotransformation of aniline and its 4-chloro
derivative [149,163] by the presence of catalase furnishes
unequivocal e vidence f or a vital role of autoxidatively
liberated H
2
O
2
in Hb-dependent catalysis. It could be
argued that Hb(III)-OOH, generated through the reaction
of endogenously released hydrogen peroxide with methe-
moglobin, might not act itself as the oxidant, but repr esent a
transient i ntermediate in the production of oxygenating

ferryl material. However, it seems improbable that the route
of ferryl-Hb formation should b e preferentially via the
Ôperoxide shuntÕ: the sluggish autodecomposition of H bO
2
(k % 10
)3
M
)1
Æs
)1
) in the presence of the anilines [166] to
finally yield H
2
O
2
together with th e relatively low rate of
peroxide association with ferric globin (k ¼ 4.8 ·
10
2
M
)1
Æs
)1
) [159] undoubtedly impose considerable con-
straints on the overall rate o f compound I formation,
whereas its direct generation upon electron introduction
into the o xyferrous en tity is a very rapid process as oulined
above [134]. The sum of these findings refutes compound I
to contribute to s ignificant extent t o the total amount of
hydrogen peroxide-induced active oxidant, but rather

favours Hb(III)–OOH itself or the iron(III)-H
2
O
2
adduct
[110] to serve in this f unction (Scheme 3). This view is
endorsed by the fact that imidazole, acting as the p roximal
axial ligand in electron-rich iron-po rphyrin model com-
pounds, appears to prolong the lifetime o f the H
2
O
2
-derived
hydroperoxo-iron species, such as to permit direct oxygen
insertion into s ubstrate [167]. I n accord w ith this, loss of the
proximal H93 ligand in Mb through replacement with
cysteine results in enhanced O-O bond scission of oxidant
produced with organic hydroperoxide [168].
Heme oxygenase. Although heme oxygenase (HO; EC
1.14.99.3) is distinct from P450s, t he reactions catalyzed by
this enzyme are, nevertheless, part of the same heme-
dependent reaction manifold that underlies the catalytic
action of all h emoproteins. The first metabolic process
mediated hy HO is self-hydroxylation of heme to form
a-meso-hydroxyheme ( Scheme 4), using the histidyl-ligated
heme group as both a prosthetic unit and substrate
[125,169–171]. Whereas transformation by plant [172] and
bacterial [ 173] HO enzymes requires electron supply b y an
NADPH-ferredoxin reductase/ferredoxin couple analogous
to mitochondrial and microbial class I P450s, mammalian

heme oxygenases accept reducing equivalents, in the
presence of dioxygen, from NADPH-P450 reductase,
resembling microsomal class II P450s with respect to their
ability to f unctionalize unactivated C-H bonds [170]. There
is strong evidence for Fe(III)-OOH to act as the meso-
hydroxylating species in HO catalysis. Thus, H
2
O
2
has been
found to be able to replace NADPH/O
2
in supporting
the first step in heme oxidation, while ferryl-forming
acyl hydroperoxides were incompetent [174]. Mo reover,
application of ethyl hydroperoxide as the o xidant could be
demonstrated to promote generation of a-meso-eth oxyheme
[175]. Studies with the four meso-methylmesoheme
regioisomers disclosed t he electron-donating methyl
substitutents to govern t he regiochemistry of meso-
hydroxylation on an electronic rather than steric basis,
implicating e lectrophilic addition of the oxygen to the
porphyrin ring [176,177]. It should be mentioned, in this
context, that the H39V mutant of rat outer mitochondrial
membrane cytochrome b
5
has been shown to be capable of
building a coordinate ferric hydroperoxo intermediate upon
reduction of the oxyferrous complex w ith h ydrazine, which
adds a hydroxyl group to the porphyrin to produce meso-

hydroxyheme [178].
Direct evidence for the occurrence of Fe(III)-OOH
during normal catalytic turnover of HO was furnished by
EPR and EN DOR experiments [179]. Radiolytic cryo-
reduction/annealing investigations to directly monitor sol-
vent and secondary KIEs, preventing masking of the latter
by interference with other reactions, revealed bond forma-
tion between the a-meso-carbon of the porphyrin moiety
Scheme 4. Proposed mechanism of HO-catalyzed conversion of hemin
to a-meso-hydroxy-heme. Thehemeunitisshowninatruncatedform.
Reproduced from [125] with permission.
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4341
and the terminal oxygen atom of the hydroperoxo entity;
this process was activated by delivery of t he second proton
by a carboxyl donor, presumably D140 [180,181]. Finally,
optical absorption and E PR measurements permitted t he
detection of a hydroperoxo intermediate derived from a
synthetic Fe(III)-porphyrin complex, with electrophilic
addition of the axially ligating O OH
–
to the porphyrin
macrocycle to yield the cationic form of meso-hydroxy-
porphyrin [182].
Microperoxidase-8 (MP8). Microperoxidase-8 is a
heme-based mini-enzyme, forming a new generation of
biomimics, obtained by two subsequen t steps of peptic
and tryptic digestion o f horse-heart cytochrome c [183]. It
consists of a residual octapeptide, with histidine covalently
attached to the ferric heme iron as the fifth ligand (Fig. 2).
The mini-catalyst has been depicted as an a ttractive model

for studying P450-type oxygen transfer reactions [184].
Thus, addition of ascorbate to MP8/H
2
O
2
-containing
reaction mixtures to block peroxidase-type radical
chemistry and, instead, induce a P450-like oxygenation
mechanism has been demonstrated to result in a drastic
diminution of polymerization products derived from
aniline and some of its p- and N-substituted congeners,
while formation of p-hydroxylated and dealkylated
metabolites was in creased; this w as attributed to involve-
ment in cataly sis of a (hydro)peroxo-iron intermediate
[185,186]. In accord with this, NADPH/O
2
-sustained
conversion of aniline to p-aminophenol by heme-peptide
reconstituted with NADPH-P450 reductase has been
shown to be highly susceptible to t he presence of
catalase [187]. Moreover,
18
O-labeling experiments with
MP8 in the presence of ascorbate revealed the biocatalyst
to p-hydroxylate aniline with full transfer of oxygen from
H
18
2
O
2

, while rapid exchange of the labelled oxygens of
H
2
O
2
with unlabelled H
2
O occurred, pointing at reversi-
bility of f ormation of the high-valent iron-oxene s pecies to
produce a porFe(III)-H
2
O
2
complex [188]. Similarly, water
has b een advocated to play a decisive role in regeneration,
throughreactionwith(R
•
+
)MP8Fe(IV) ¼ O, of the active
oxidant operative in the microperoxidase/H
2
O
2
-driven
hydrocarbon oxygenation in bi- and tricyclic aromatic
compounds [189] or oxidative aromate dehalogenation
[185,190] ( Scheme 5). It should be emphasized t hat
reaction of iodosylbenzene with purified CYP2B1 [191]
or nonporphyrin iron(III) chelates in basic media [132,192]
has b een detected to prompt O-O bond formation a t the iron

centres. Alkoxylating dehalogenation of halophenols,
carried out by MP8/H
2
O
2
in alc oholic solvents, has been
hypothesized t o implicate a mechanism, in which the iron-
oxene resonance form reacts with alcohol to generate an
Fe(III)-OOR intermediate [193]. Unequivocal indenti-
fication of the MP8-based (hydro)peroxo-iron(III) entity
has been achieved by optical absorption [194] and rapid-
freeze EPR measurements [195].
Synthetic metalloporphyrin models. Synthetic metallo-
porphyrins (Fig. 3) were selectively tailored as models of
the P450 active site to gain more detailed i nsight into the
mechanistic b asis of oxygen transfer reactions. Using a set
of meso-tetraarylporphyrine derivatives (Fig. 3A), cis-
stilbene was found to be subject to H
2
O
2
-sustained
conversion to the oxide metabolite in aprotic solvent w ith
trace amounts o f allylic oxidation products, ruling out iron-
oxene or OH radicals to be responsible for olefin
epoxidation, while hydroperoxo-iron was likely to be
the active oxidant [124,167]. S imilarly, the lack of
18
O-incorporation, at low temperature, from labeled water
during perbenzoic acid-supported epoxidation of

cyclooctene by the polyhalogenated TPFPP analogue was
interpreted to mean that the electronegatively substituted
iron porphyrin generated a relatively stable Fe(III)-OOR
species, w hich directly transferred its oxygen to t he olefin
[123]. This postulate i s in line with the observation that there
is a strict relationship between the selectivity of
norbornylene over a-methylstyrene epoxidation by the
TDCPP-porphyrinato-iron complex and the structure of
the peracids u sed [196]. The same principle also applies to
the cyclooctane vs. cyclooctene oxidation catalyzed by a
thiolate-ligated meso-tetraarylporphyrin model (Fig. 3B)
with various p-substituted perbenzoic acids [197].
Fig. 2. Ch emical structur e of microperoxidase-8. Data taken from
[190].
F
NH
H
3
C
N
H
3
C
OHF
H
2
O
H+
F–
N

H
3
C
O
Por – Fe
III
– OOH Por – Fe
IV
– O
Por – Fe
III
– OOH
Por – Fe
III
– OH
Scheme 5. Proposed reaction mechanism for a MP-8 -catalyzed dehalo-
genation pathway.
4342 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
Moreover, acylperoxo-iron(III) has been claimed to also
serve as the effective catalyst in the peracid-dri ven
cyclohexane hydroxylation depending on the n ature of the
anionic axial ligands of the Fe(TPFPP) adduct [198].
Studies with a second genre o f metalloporphyrin systems,
containing molecular oxygen and a coreductant such as
ascorbate to m imic the natural P450-mediated electron
transfer pathway, disclosed manganese meso-tetraphenyl-
porphyrin to substantially differ from the iodosylbenzene-
promoted route with respect to regioselectivity of olefin
epoxidation and reactivity toward tertiary vs. secondary
C-H bonds; this was tentatively attributed to the involve-

ment of a Mn(III)-peroxo instead of a ferryl c omplex in the
ascorbate/O
2
-driven process [199].
The r eactive (hydro)peroxo-metalloporphyrin species in
the a bove m odel s ystems have been charact erized b y visible
spectroscopy, NMR, EPR, and Mo
¨
ssbauer data upon
combination o f the tetraphenyl- or octaethylporphyrin-
metal adducts (Fig. 3D) with superoxide anion [129,
200,201] or reduction of oxygenated macrocycle by ascorbic
acid [202]. Using the methylmercaptane porphyrin model
depicted in Fig. 3C, the optimized geometr y of the transient
reduced ferrous dioxygen form, calculated by applying
nonlocal DFT methods, indicated an a symmetric Ôend-onÕ
binding fashion of the dioxygen ligand with pronounced
elongation of the Fe-O and Fe-S bonds [203].
Evidence from comparative studies with mononuclear
nonheme iron enzymes and biomimetic metal chelates
Rieske-type dioxygenases. Rieske oxygenases catalyze
the r egio-and stereospecific cis-dihydroxylation of a romatic
rings, in itiating aerobic degradation of aromatic compounds
in soil bacteria, and are targets for bioengineering in
bioremediation [204]. They a re a consortium of two o r
three protein components involving a Rieske Fe
2
S
2
cluster to

channel electrons from N AD(P)H via a flavin-containing
reductase to a mononuclear iron ce ntre; the latter is believed
to b e the site of d ioxygen and substrate activation [204]. This
electron transfer chain thus functions like the heme centres in
class I P450s acting in unison with their associated iron-
sulfur redox partners [135]. Analogous to the role of
putidaredoxin as an effector in CYP101 catalysis [50,67],
binding of phthalate oxygenase reductase (EC 1.18.1), a
flavo-iron-sulfur polypeptide, to phthalate dioxygenase
(PDO; EC 1.14.12.7) has been advocated to tune the
enzyme’s structure for oxygenating activity on an allosteric
basis [205]. Moreover, toluene dioxygenase (TDO;
EC 1.14.12.11) and naphthalene 1,2-dioxygenase (NDO;
EC 1.14.12.12) have been found to mediate P450-like
monooxygenations when provided with appropriate
substrates [206,207].
Availability of the crystal structure of NDO as well as
spectroscopic data provide a rationale for the catalytic
mechanism of this class of enzyme s. Naphthalene 1,2-
dioxygenase is a heterohexamer composed of an equimolar
combination of a-andb-subunits, each a-subunit bearing
an Fe
2
S
2
cluster and a mononuclear iron site [208]. Two
histidines and one bidentate aspartate ligand, the socalled
Ô2-His-1-carboxylate facial triadeÕ, encountered with var ious
nonheme iron, oxygen-activating enzymes, occupy one side
of the mononuclear iron coordination sphere [209]. Sub-

strate binding to produce a n open c oordination position o n
Fe(II) has been suggested to be critical in O
2
-activation,
allowing two-elctron transfer from both the mononuclear
iron centre and the reduced Rieske cluster to g enerate an
Fe(III)-OO(H) intermediate [210,211]; the latter has been
proposed to exert a concerted m ode of attack on substrate,
explaining the cis -specificity of the dihydroxylation reaction.
In accord with this concept, hydrogen peroxide has been
reported to be able of substituting for NAD(P)H/O
2
in
NDO-dependent cis-dihydrodiol formation, both oxygen
atoms in the product deriving p rimarily from H
2
O
2
[212].
Moreover, ben zene h as been demonstrated to act as both a
substrate and an uncoupler of NDO, causing t he release o f
H
2
O
2
during the reaction [ 213]. M ore r ecently, the role of a
putative end-on (hydro)peroxo-iron catalyst in NDO
turnover has been ascertained by the detection, in the
enzyme’s crystal structure, of an indole-oxygen adduct
bound to the m ononuclear iron [214]. C ircumstantial EPR

N
N
N
N
R
R
R
R
Fe+++
Me
Me
Me
Cl
Cl
F
F
F
F
F
F
F
N
N
N
N
F
e
+
++
HN

O
O
S
N
N
N
N
Fe+++
Et
Et
Et
Et
Et
Et
Et
Et
Cl
–
TDCPP
TPFPPTDFPP
S
H
H
H
A
B
C
D
R=
TMP

Fig. 3. Ch emical structures of iron(III)por-
phyrin co mplexes used as P450 m odel species.
Data ta ken from [167,197,202, 203].
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4343
spectrometric and solvent isotope effect studies with 4-
methoxybenzoate O -demethylase, a two-component system
comprised of a flavo-iron-sulfur reductase and putidamono-
oxin (PMO; EC 1.14.99.15) as the t erminal oxygenase, lent
further support to the idea of peroxo-iron-sustained
oxygenation chemistry [215,216]. Apart from O-demethy-
lation, PMO can functio nalize aliphatic and aromatic C-H
bonds, with H
2
O
2
being liberated in the presence of
uncoupling c ompounds [217,218]. By a substrate-modulated
reaction, PMO has been demonstrated to also act as a
peroxotransferase: using v inylbenzoate as the substrate, the
enzyme was found to form 4-(1,2-dihydroxyethyl)benzoate
with both oxygen atoms being incorporated into the
product from atmospheric
18
O
2
[218]. This metabolic
pattern might reflect ring opening of an epoxide inter-
mediate at either of its two C-O bonds [219,220].
The sum of these findings strongly invokes the notion
of oxygen activation during redox cycling of the diverse

dioxygenases to p roceed along a common track, with
Fe(III)-OO(H) serving a s t he preponderant oxidant.
Clearly, some contribution to catalysis by high-valent
iron-oxene cannot be dismissed [211,221]. Scheme 6 out-
lines the putative pathway of PDO-dependent cis-dihyd-
roxylation as proposed previously [222,223]. Credibility of
the mechanistic scheme is e nhanced by results obtained
with iron-based f unctional models for Rieske dioxyge-
nases. Introduction into the ligand frameworks depicted in
Fig. 4 of more than one 6-methyl substituent to modulate
the electronic a nd steric properties of the ligand e nviron-
ments has been recognized to afford high-spin hydroper-
oxo-iron species in combination w ith H
2
O
2
, exhibiting
strong predilection for cis-dihydroxylation of olefins at the
expense of epoxidation [224,225]. A side-on Fe(III)-OOH
entity, generated by isomerization of its end-on congener,
or the cis-iron(V)-oxo(hydroxo) valence tautomer have
been implicated as alternative catalysts in the dominant
cis-diol formation from alkenes. However, evidence for
participation in these reactions of the high-valent oxidant
seems e quivocal in view of the nearly insignificant amount
of
18
O-incorporation from water into the diol products
[225].
Bleomycin and related metal-based model complexes. The

bleomycins (BLMs) constitute a f amily of natural
glycopeptide antibiotics produced by the fungus
Streptomyces verticillus, which are used as antineoplastic
agents owing to their ability to degrade DNA upon
bioactivation i n t he presence of appropriate metal ions
and a source of dioxygen [226]. Although iron appears to b e
the most effective BLM cofactor, other metals also bind
strongly to the antibiotic [227]. A key to the unique
reactivity of the nonheme iron(II) site of BLM (Fig. 5)
toward O
2
seems t o r eside i n one of its equatorial ligands,
the pyrimidine m oiety [228]. T he mechanism of oxygen
activation is strongly reminiscent of P450 cycling [226],
the f erric intermediate being reduced eithe r chemically by
dithiothreitol and ascorbate [229] or enzymatically by
NADPH-P450 reductase [230]. Oxygen surrogates such as
iodosylbenzene [229], H
2
O
2
or alkylhydroperoxides [231]
have been shown to be apt to bypass reductive
Fe++ Fe++ Fe
3
+
O
O
Fe
3

+
O
O–
Fe
3
+
OO
Fe
3
+Fe
3
+
COO––OOC
COO––OOC
HO OH
H
H
COO––OOC
COO––OOC
COO––OOC COO
–
–OOCCOO––OOC
O
H
H
O
(H+)
2 H+
2 e-
from

reductase
2–
R
red
R
red
R
red
R
ox
R
ox
R
ox
R
ox
+ O
2
products
or
Scheme 6. Possible molecular mechanism for a
PDO-promoted cis -dihydroxylation r eaction.
R
red
and R
ox
denote reduced and o xidized
Rieske c entre, r espectively. Reproduced f rom
[223] with permission.
Fig. 4. Iron-bound ligand fram eworks used as models of dioxygenases.

Data taken f rom [224].
4344 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
O
2
-activation. The architecture of ac tivated BLM has b een
unequivocally demonstrated to be consistent with an end-
on, low-spin Fe(III)-OOH intermediate [231–235], tightly
organized through H-bonding of the peroxide unit to the
threonine side chain of BLM [236]. In accord with this,
superoxide anion can participate in activated-BLM
formation [237,238]. The accepted mode of reactivity
toward DNA is hydrogen atom abstraction from the C4¢
position o f the DNA deoxyribose s ugar, for which multiple
scenarios have been set f orth including hetero- or homolytic
O-O bond cleavage of activated BLM to yield a high-valent
oxo-iron sp ecies affording DNA attack, or d irect re action of
HOO-Fe(III)BLM with its targe t t o give a DNA radical,
H
2
O, and an Fe(IV) ¼ O entity [226,227,239,240]. Among
these possibilities, the homolytic pathway, entailing the
production of undiscriminating free OH r adicals, c ould be
discounted in view of the high selectivity of DNA strand
breakage [239]. Also, density functional theory (DFT)
calculations of the electronic structure of an optimized
geometric model of activated BLM predicted the heterolytic
mechanism t o be energetic ally unfavourable by at least
40 kca lÆmol
)1
, w hich is more than 150 kcalÆmol

)1
less like ly
than for P450 [223,241]; this wa s attributed to differences in
the n ature o f the axial anionic ligands. These observations
tend to favour direct participation of hydroperoxo-iron
in catalysis, a reaction considered to be approximately
thermoneutral. Calculations revealed protonation of the
peroxo precursor t o considerably increase electrophilicity of
the oxidant [223,241]. Experiments with HOO-Co(III)BLM
ÔgreenÕ, a stable analogue of activated BLM, p rovided a rare
snapshot of a reactive intermediate poised to initiate the
hydrogen atom abstraction event: t he distal oxygen of the
hydroperoxide is only 2.5 A
˚
away f rom the C4¢-H of
cytosine [242]. Further i nformation was gained from studies
with synthetic iron complexes assumed to be model system s
for BLM because of their capabilities to inflict DNA strand
scission in the p resence of ascorbate/O
2
[192,243]. The
process was shown to be sensitive to the action of
superoxide dismutase or catalase, implicating the
involvement of a peroxo adduct in catalysis [243]. In fact,
reactivity was enhanced when H
2
O
2
was the oxidant in p lace
of reductant and air [243], and the putative Fe(III)-OO(H)

intermediate could be characterized by spectroscopic
techniques [244].
Apart from DNA degradation, activated BLM can
promote P450-like monooxygenations with low-molecular
substrates such as ring hydroxylation of aromatic com-
pounds [233], N-dealkylation of arylamines [229], or epoxi-
dation of alkenes [ 229,236,245]. Noteworthy, experiments on
BLM-mediated epoxidation of cis-stilbene, employing iod-
osylbenzene as the oxygen donor in the presence of H
18
2
O,
disclosed the epoxide oxygen to derive primarily from labeled
water [229,245]. As pre-equilibration of the oxidant with
water prior to activation has been demonstrated not to result
in exchange with H
2
O [229,246,247], this observation
strongly suggests O ¼ Fe(IV)BLM
•
+
to be capable o f
undergoing exchange with solvent to regenerate Fe(III)-
OOH as t he epoxidizing species [132]. This view is substan-
tiated by studies with the iron complex of the BLM mimic
depicted in Fig. 6A. Here, iodosylbenzene in basic media
gives rise t o formation of a hydroperoxo intermediate
presumed to be the catalyst in the oxidative turnover of
olefins [192]. Hydrogen abstraction from methanol, used as
the solvent, to produce a radical intermediate has been

ascertained to result from direct reaction with the alcohol of
the hydroperoxo unit formed by c ombination of H
2
O
2
with
the iron-ligated BLM model shown in Fig. 6 B [248]; the
methoxy radical thus generated has been found to induce
ligand modification through attack o n the carbon a to the
amidate.
A plethora of bioinspired ferric ( hydro)peroxo complexes
with the peroxide ligand being bound in a side-on (g
2
)or
end-on (g
1
) fashion have been cr eated as more general
models of metalloe nzymes by r eacting H
2
O
2
or ROOH with
various iron chelates [223,249]. While the side-on peroxo
Fig. 5. Ch emical structure of iron b leom ycins.
Data taken f rom [240].
NH
NH
HN
HN
N

N
N
N
N
Br
H
N
H
N
NH
2
N
NH
O
N
H
O
N
N
N
N
N
N
N
NN
N
N
NN
R
2

R
1
R
2
1: R
1
=H; R
2
=Me
2: R
1
=R
2
=Me
A
B
C
F
E
D
Fig. 6. Nitrogenous ligands used to construct the corresponding iron
complexes as mimics of b leomycin and m ononuclear nonheme metallo-
enzymes. Data taken from [ 192,248,252,254].
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4345
entity is relatively inert t oward organic substrates such as
alkanes and alkenes, protonation to increase electron
affinity has been recognized to be a means of generating a
highly reactive species [250,251]. However, in most cases
available data are insufficient to unravel the intricate
mechanism of oxygen transfer. Judging from the limited

number of reports based on isotope labeling and kinetic
studies, hydroperoxo-iron or its alkylperoxo analogue can
act as direct oxidants in hydrocarbon hydroxylation [252],
alkene epoxidation [253,254], and alcohol oxidation
[122,255] promoted by iron-bound members of the pyrid-
ine/amine ligand family presented in F ig. 6 C–F. Similarly,
Cr-OOH, produced with a macrocyclic chromium-based
system (Fig. 6C), exhibits H
+
-assisted o xidative reactivity
toward triarylphosphines [256]. Generally, t he peroxide-
driven route appears to be under stereochemical control
exerted by a-subst ituents [252] on the pyridyl moiety
(Figs 6D,E) or topological constraints [253] imposed by
the isomeric nature of the ligands involved (Fig. 6F).
Finally, combination, i n the p resence of atmospheric
dioxygen, of a relatively labile Fe(II) complex such as b is
(2,2¢-bipyridine)iron(II), bis(picolinate) iron(II), or bis(dip-
icolinate)iron(II) with a reductant such as diphenylhydra-
zine to constitute a so-called Mimoun system [257] has been
shown t o provide a useful tool to assess the molecular basis
of alkane [258,259] and phenol [260] hydroxylation. The
mechanistic scheme for such reactions embodies the occur-
rence of a ternary catalyst/reductant/O
2
adduct [258]
analogous to P450 chemistry, so that more concerted r edox
steps can be envisioned. Substrate transformation posits the
participation of structurally similar h ydroperoxo-iron cat-
alysts with different f ormal o xidation states [258,260], the

process being driven to exothermicity via water formation
(Eqn 3).
RH þ PhNHNHPh þ O
2
! ROH þPhN ¼ NPh þH
2
O
ð3Þ
Electrochemical investigations support this concept,
revealing autoxidation of diphenylhydrazine, when
exposed to O
2
, to liberate hydrogen peroxide, which
collapses with iron(II) to give an Fe(II)-H
2
O
2
complex
directly leading to metabolic turnover [261].
Peroxo-iron acting as a nucleophilic oxidant
in P450-catalyzed hydroxylations
Evidence from kinetic analysis of P450 function
Steroidogenic P450s belong to the category of isozymes
promoting multifunctional biosynthesis of endogenous
compounds. Thus, 17a-hydroxylase-17,20-lyase (CYP17)
sustains conversion of pregnenolone/progersterone to
androstenediol/androstenedione via primary attack on the
17a-position of t he pregnene nucleus, followed by o xidative
acyl-carbon cleava ge of the 17a-hydroxy intermediate(s)
formed to eject acetate [262]. Incubation of microsomal

fractions prepared from pig testes with deuteriated preg-
nenolone under an atmosphere of
18
O
2
permitted analysis o f
the pattern of isotope incorporation into the reaction
products, best rationalized by invoking the participation of
a nucleophilic pe roxo-iron spec ies in the C-C bond fission
process [263,264]. It has been hypothesized that 17a-
hydroxy-progesterone binds to unprotonated CYP17 such
as to interrupt a p roton shuttle to Fe(III)-OO
–
, facilitating
C-17 side-chain dissociation through peroxide chemistry
[265]. Employing r ecombinant CYP17 and labeled h ydroxy-
androstene-17b-carbaldehyde, a p regnenolone analogue, a s
the substrate in combination with
18
O
2
, isotope-partitioning
experiments suggested androgen genesis to b e closely linked
to formation of an iron-peroxy adduct prone to fragmen-
tation [266]. A similar p aradigm also appears to a pply to the
final step in aromatase (CYP19)-catalyzed biotransforma-
tion of androgens to estrogens [267]. As illustrated in
Scheme 7, this event entails aromatization of the A-ring
of androstenedione via oxidative decarbonylation of the
19-aldehyde intermediate to release formic acid with the

concomitant production of estrone [268]. When human
placental microsomes fortified with deuteriated androgen
precursors in the presence of
18
O
2
were used to explore the
mechanistic course of aromatizatio n, a transient ferri
peroxy-hemiacetal-like complex (Scheme 7) turned out to
be a strong contender to explain the step of C-C bond
scission [269,270]. This type of nucleophilic attack on a
carbonyl group by peroxo-iron has also been evidenced in
the final C-C bond cleaving process during sterol biosyn-
thesis in the lanosterol 14a-demethylase (CYP51) reaction
cascade [271,272].
Of particular interest, the NADPH/O
2
-dependent con-
version of cyclohexane carboxyaldehyde to cyclohexene by
reconstituted CYP2B4 has been evaluated as a potential
model for deformylation reactions brought about by
steroidogenic P450s: mass-spectral analysis unveiled f or-
mate to be formed in about an equimolar amount with
respect to olefinic product [11]. Similarly, a series o f other
xenobiotic C-5 aldehydes have been shown to be deform-
ylated to variable extent by h ighly purified rabbit liver P450s
[273]. Moreover, externally added H
2
O
2

in place of the usual
O
2
-reducing system has been reported to be active with
CYP2B4 in supporting deformylation of aldehydes [11,274].
Employing 3-phenylpropionaldehyde as the substrate, an
adduct was detected with a m ass corresponding to that of
native heme modified by a phenylethyl group, presumably
arising from the reaction of a peroxo-iron entity with the
aldehyde to give a peroxy-hemiacetal [274].
Evidence from experiments with genetically engineered
P450 enzymes and molecular modelling
Taking advantage of the debilitating effect on proton
delivery to Fe(III)-OO
–
exerted by m utagenesis of t he highly
conserved active-site threonine in P450s (see above),
experiments conducted with the T306A mutant of CYP17
disclosed an about sevenfold increase i n the proportion of
acyl-carbon cleavage vs. hydroxylation activity during
androgen biosynthesis as compared with the wild-type
enzyme [275]. T his was t aken to substantiate the assumption
of juxtapositioning of the nucleo philic ferric peroxide anion
and t he carbonyl g roup of the substrate to b e a compulsory
prerequisite for directing the enzymatic flux toward C-C
bond rupture. Similarly, replacement of glutamate at
position 302 in the CYP19 polypeptide with residues such
as alanine or valine proved to be deleterious to conversion
of androgens to estrogens [276]. Based on an active site
4346 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004

model constructed by alignment of the CYP19 sequence
with the known crystal structures of bacterial P450s, E302
was postulated t o be essential to activation of the 1 9-oxo
group of the s ubstrate f or attack by the peroxo-iron species
[268,277], with D309 playing an important role in the
aromatization process in concert with a histidine residue
through facilitating abstraction of th e 2b-hydrogeninthe
A-ring of the C-19 substrate and donation of a p roton t o the
3-keto entity, respectively, to permit enolization [ 277,278]
(Scheme 7 ). A Ôthreonine switchÕ, conferring regulatory
function on the conserved threonine-310 during peroxo-
iron-mediated aromatization has been proposed, though
experimental results obtained with the T310S variant of
CYP19 w ere a mbiguous [277]. In f act, switching from i ron-
oxene to peroxo-iron chemistry t hrough threonine-302 to
alanine mutagenesis of truncated CYP2B4 could be dem-
onstrated by studies comparing the catalytic specificity o f
deformylation o f cyclohexane carboxaldehyde with t hat of
hydroxylation of other compounds [279]. Moreo ver, inves-
tigations on the mechanism-based destruction of CYP2B4
by aldehydes revealed a ugmented inactivating potency with
the T302A congener, emphasizing the notion of a kinship
to aldehyde deformylation via a peroxyhemiacetal inter-
mediate [280].
Evidence from comparative studies with non-P450
hemoproteins and metalloporphyrin models
Nitric oxide synthase. Nitric oxide synthases (NOS;
EC 1.14.13.39) comprise a family of thiolate-ligated
constitutive or inducible hemoprotein isoforms [281],
exhibiting insign ificant sequence identity with P450s in

the heme-binding region [282], but bearing a C-terminal
flavoprotein fragment in the single polypeptide chain
structurally resembling NADPH-P450 reductase; the latter
is separated from the heme domain by a calmodulin
consensus binding sequence [283]. Importantly, NOS
enzymes are dimeric proteins, in which flavin-to-heme
transfer of electrons provided by NADPH proceeds
exclusively between adjacent subunits in the heterodimer,
implying domain swapping for proper alignment of the
reductase and oxygenase entities [284]. Tetrahydrobiopterin
(BH
4
), located close to the heme unit [285], has been shown
to contribute to s tabilization of the NOS dimers [286].
Moreover, the modifier binds cooperatively to the substrate-
binding region [287] and facilitates electron flow to
oxyferrous NOS [288].
The physiological role of NOS pertains to t he pro duc-
tion of NO
•
, a n important signaling molecule, and c itrulline
through oxidative degradation of N
G
-hydroxy-
L
-arginine,
generated via primary N-hydroxylation o f one of the two
equivalent guanidino nitrogens of arginine [281]. While
L
-arginine and the homo-

L
-arginine derivative have been
originally thought to be the only true N OS substrates,
more recent stu dies unveiled a series of N-aryl-N¢-hydroxy-
guanidines to serve as N O
•
donors after oxidative activa-
tion [289]. Circumstantial analysis of the stoichiometry of
the N O-forming reaction d isclosed a three -electron process,
with decomposition of the N-hydroxyarginine intermediate
consuming only 0.5 equivalents of NADPH per mol of O
2
during nitroxyl radical ejection [290]. Comparative studies
with microsomal P450s [291,292] and biopterin-free as well
as BH
4
-containing NOS [293], exhibiting product s electiv-
ity with respect to the almost exclusive, superoxide
dismutase-insensitive generation o f equimolar amounts o f
urea and NO
•
from arginine and some non-a-amino-acid
Scheme 7. Postulated final oxidation s tep in
aromatization catalyzed by CYP19. Data
adapted f rom [268] w ith permission.
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4347
N-hydroxyguanidines, were designed to unravel the puz-
zling stoichiometric behaviour. The results from these
investigations support a unifying concept, predicting the
conversion of N-hydroxyarginine to citrulline a nd NO

•
to
be mediated by a peroxo-iron catalyst, generated via the
donation of one electron by the N-hydroxy intermediate to
oxyferrous NOS, formally [Fe(III)-O
2
•
)
]. The F e(III)-OO
–
complex derived from this reaction would undergo nucle-
ophilic addition to the C ¼ N bond of the substrate cation
radical to initiate decomposition of the adduct with the
elimination of NO
•
[289,294,295]. A modified version of
this mechanism ( Scheme 8), developed on thermodynamic
reasons, envisages deprotonation of N-hydroxyarginine to
yield hydroperoxo-iron as the oxidant and an iminoxyl
radical. Nucleophilic attack of the Fe(III)-OOH species on
the substrate radical triggers fragmentation of t he complex
to relea se N O
•
[295–297]. There is evidence for a r egulatory
function of BH
4
in NO
•
formation from N-hydroxyargi-
nine [293,298], however, t he precise mechanism of t his

action remains obscure. On the whole, the second step in
NOS catalysis closely resembles aromatase chemistry as
detailed above.
Chemically modified myoglobin (rMb). Although
native Mb has been demonstrated to ex hibit P450-like
monooxygenase activity when combined with an
appropriate electron-transport system [143], efforts were
undertaken to engineer the catalytic properties of M b
through functionalization of the pigment by c hemical
modification of its prosthetic heme unit [299]. Thus,
studies on the NADH-driven deformylation of 2-phenyl-
propionaldehyde by rMb, reconstituted with an electron-
accepting isoalloxazine (flavin) moiety covalently a ttached to
one heme propionate, revealed acetophenone to be the
unique product [300]. A similar metabolic pattern was
observed when t he carbonyl group of the secondary
aldehyde was subject to nucleophilic attack by biomimetic
peroxo-iron(III) porphyrin c omplexes [301] (see b elow).
This parallelism was thought to be indicative of the
participation in the rMb-dependent deformylation
pathway o f a nucleophilic Fe(III)-OO
–
catalyst [300]. While
1-phenylethanol has been detected to be a further metabolite
derived from 2-phenylpropionaldehyde during o xidative
deformylation by CYP2B4 [280], the lack of alcohol
product in the rMb-promoted reaction was reasoned to
arise f rom competition between oxygen rebound and PhC
Æ
HCH

3
quenching by dissolved O
2
, as formation of the radical
intermediate is believed to occur near the surface of the
flavohemoprotein c onjugate [ 300].
Synthetic metalloporphyrin models. Peroxo-iron(III)
derivatives, coordinated in a side-on (g
2
)geometry,were
created by reacting synthetic porphyrin complexes with
potassium superoxide in aprotic solvents, yielding
oxygenating model systems for comparison with P450
chemistry. Such studies have identified certain ferric peroxo
porphyrins as remarkably strong nucleophiles capable
of oxidizing a variety of electron-deficient molecules
[302]. In this way, [Fe(III)(TMP)(O
2
)]
–
(Fig. 3 A) and
[Fe(III)(PPIXDME)(O
2
)]
–
, t he peroxo adduct of the
dimethyl ester of protoporphyrin IX, were found to
epoxidize electron-poor olefins such as 2-cyclohexen-1-one
or 2-methyl-1,4-naphthaquinone (menadione) via direct
oxygen insertion into an olefinic bond (Scheme 9A),

wereas no reactivity was observed toward electron-rich
organic substrates such as tetramethylethylene or tri-
phenylphosphine [303,304]. Moreover, the particulatly
electron-deficient, perfluorinated peroxo species [Fe(III)
(F
20
TPP)(O
2
)]
–
(Figure 3 A) did not show interaction with
menadione when metabolic turnover was assessed in
acetonitrile solution [303,304]. Surprisingly, epoxidation o f
the o lefin was switched on, when the reaction was allowed t o
proceed in neat dimethyl sulfoxide, a solvent capable of
axial ligation [305]. Generally, comparative investigations
with iron(III)-, manganese(III)- and titanium(IV)-
based metalloporphyrin peroxo complexes in the presence
of electron-poor olefinic substrates revealed the ferric
peroxo conge ner to be by far the most nucleophilic
oxidant [306].
Ferric peroxo porphyrins were also employed as surro-
gates to decipher the molecular events in t he final s tep of
estrogen biosynthesis by CYP19. Initial studies demonstra-
ted [ Fe(III)(TMP)(O
2
)]
–
and structurally re lated peroxo
complexes to be m ediators of C-C bond scission in aliphatic

aldehydes [ 301], mimickin g the first part of the aromatiza-
tion process. The above peroxo species was also found to be
able of rapidly r eacting w ith a n enolized analo gue of the A
and B rings of androstenedione [307] to generate aromatized
product with the ejection of formate (Scheme 9B). This
observation is in accord with the capacity of H
2
O
2
to slowly
Scheme 8. Proposed mechanism for the second
step in NOS-dependent convers ion of N-hy droxy-
arginine to citrulline and NO. Reproduced fro m
[297] with p ermission.
4348 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
react with the enolized version of the natural substrate to
yield the corresponding estrogen derivative [308].
Conclusions and future prospects
While the consensus view of the P450 mechanism, implica-
ting a high-valent iron-oxene species as the predominant
catalyst in a host of c rucial biological oxidations, h as a great
deal of weight, accumulating evidence points a t the parti-
cipation in substrate turnover of alternative a ctive oxidants
such as (hydro)peroxo-iron intermediate(s). This notion is
partly endorsed by isotope part ition experiments [99–102],
though it must n ot be disregarded that, with a branched
reaction sequence yielding m ultiple products from the same
P450-substrate complex, isotopically sensitive Ômetabolic
switchingÕ may p erturb the product distribution pattern
without a change i n the basic oxygenating mechanism [309].

Moreover, the involvement of cationic intermediates in
some aspects of P450-catalyzed hydroxylations needs
unambiguous interpretation. Whereas this scenario was
regarded to be compatible with OH
+
insertion b y hydro-
peroxo-iron into a C-H bond [9,107], carbocation formation
trough radical oxidation was envisaged as an alternative
possibility [310,311]; the latter route w ould require that
oxidation reactions proceed at higher rates than usual
radical rearrangements [98,312]. On the other hand, the
timing of radical rearrangement (radical clocks) may depend
critically on the tightness of the radical cage and the
ensemble of steric and electronic forces experienced by the
incipient radical within the variable cage [313]. For
substrates with a very strong C-H bond and a small steric
size, both effects w ould push t he reaction coordinate toward
a tighter radical c age with drastic shortening of the apparent
radical lifetime. This might persuade one in to questioning
the existence of a radical pat hway [106,107].
A step forward in the analysis of Fe(III)-OO(H) as a
potential catalyst was thought to be offered by the use o f
mutated P450s, bearing alanine or some other amino acid in
place of t he highly conserved active-site threonine; the latter
residue is believed to be the direct proton donor to the i ron-
linked oxygen. However, generalizations as to this hypo-
thesis should b e avoided. T hus, replacement o f T252 in the
CPY101 polypeptide with O-methylthreonine gave a variant
that wa s identical to the wild-t ype enzyme in its catalytic
properties [137]. Moreover, with certain P450s such as

CYP107 [314] o r C YP152 [102] there is lack of a conserved
threonine near the putative O
2
-binding site. This opens the
possibility that threonine mutagenesis rather induces a
compensatory change in the organized, H-bonded network
of water mole cules to facilitate proton delivery [14,51]. In
fact, the T252A mutant of CYP101 displays dist ortion of
the geometry of the immediate heme vicinity: the I -helical
ÔkinkÕ seen in the w ild-type enzyme is still apparent, but the
centre o f this feature is shifted one residue toward the
N-terminus in the engineered hemoprotein [315]. Similar
observations wer e mad e with the CYP102 congener [47]. In
accord with these findings, threonin e mutation elicits a
marked change in the apparent K
d
values for t ype II
interaction of nitrogenous ligands such as metyrapone or
phenylimidazole with CYP 1A2 [316] or type I spectral
binding of pregnenolone to CYP17 [275]. Noteworthy,
some experiments were c onducted with T 302A-mutated
CYP2B4 b earing additional deletion of the NH
2
-terminal
signal anchor sequence [139,142,279,280]. Truncation of
CYP2B4, as such, has b een demonstrated to significantly
distort t he structural in tegrity of the polypeptide, s uch as to
cause tyrosine(s) and the invariant C-helical W121 residue,
aligning with W96 in CYP102, to become buried [317]. I n
agreement with the well-known function of these amino

acids in productive contacts with redox partners and
iron spin-state modulation [318], respectively, shortened
CYP2B4 displayed s everely compromised electron accept-
ance from NADPH-P450 reductase and cytochrome b
5
,
ostensibly arising from disruption of e vents involved in
second-electron transfer t o oxyferrous P450, while stability
of the active oxy-complex, once formed, remained u naffec-
ted; overall enzyme a ctivity was shown to be substantially
harassed [317]. The sum of these findings suggests extreme
caution in interpreting data obtained with t he genetically
engineered P450s, because changes in the pattern of product
distribution, as observed with various diagnostic probes,
cannot be savely ruled out to be the direct result of subtle
alterations in active-site conformation.
Extensive approaches to a better understanding of the
diversification of the oxygenating pathways focus on
comparative studies with natural and bioinspired P450
mimics uniformly bearing nitrogen-chelated iron in their
active sit es. While oxygen binding and activation in these
congeners appears t o obey i dentical rules, the specific steric
and electronic features of the iron coordination environ-
ments, no doubt, are decisive in steering the equilibrium
between metal-oxo-/metal-peroxo-driven oxygenating activ-
ity, thus modulating the pattern of product distribution
[64,168,224,253,305]. Despite this diversity, all the systems
examined carry out P450-like o xidations, i n t he presence of
both dioxygen and peroxides, obviou sly using (hydro)pe-
roxo-iron as the common catalyst. As P450s are part of this

manifold of oxygenating metalloenzymes, it seems permis-
sive to disclose parallels to their biomimics, reinforcing the
contention that Fe(III)-OO(H) might be operative as an
alternative oxidant in P4 50 cycling, too.
Novel concepts have been advanced to harmonize the
partly conflicting data on the role of multiple oxidants in
P450 chemistry. Based on the ability o f H
2
and methane to
inhibit hydrocarbon oxygenations by P450 models, an
agostic r-complex between probe a nd the iron-oxene centre
was p roposed to rearrange in c ompetition with consumma-
Scheme 9. Epoxidation of menadione (A) and deformylation of an
enolized mimic of the A/B ring moiety of androstenedione (B) by
[Fe(III)(TMP)(O
2
)]
–
. Data reproduced f rom [302] with permission.
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4349
tion of the hydroxylation reaction [319]; however, investi-
gations with a s et of alkylcyclopropanes a nd CYP2B1
provide little support to this idea [320]. Using density
functional theory, a viable Ôtwo-state reactivityÕ (TSR)
paradigm has been delineated applying to the intermediate
iron-oxo species [321–324]. The model e ntails competition
between a spin-paired low-spin ensemble and a nonspin-
paired high-spin manifold, with state crossing being asso-
ciated with either an insertion or a radicalar pathway,
permitting the formation of cationic derivatives through

radical oxidation. Spin inversion should be probe depend-
ent. The proposed mechanistic scheme has a certain
intellectual attrac tion in s atisfactorily explaining co ntrover-
sial P450 data [311,325], especially those p ertaining to the
unusually high rates of oxygen rebound met with hyper-
sensitive radical clocks [103,104,320]. The theory has also
been used to characterize alkene epoxidation [326,327].
Scheme 10 depicts the potential energy surface for ethene
epoxidation. This reaction can b e brought about in a
synchronous or asynchronous mode, the latter being
energetically more favourable. The activation energies for
the low-spin (
2
TS1) and high-spin (
4
TS1) states in the
asynchronous route are 14.9 and 13.9 kcalÆmol
)1
, respect-
ively. While collaps of the quartet high-spin t ransition states
(
4
2-III and
4
2-IV) to the corresponding high-spin, iron-
coordinated epoxy p roducts exhibits an energetic b arrier of
7.2 and 2.3 kcalÆmol
)1
, a tiny barrier of < 0.3 kcal Æmol
)1

permits conversion of the doublet low-spin intermediates
(
2
2-III and
2
2-IV) to the epoxide c omplexes. The low energy
required f or transformation of the transition s tates in the
asynchronous pathways, making them stepwise but appar-
ently concerted, has been advocated to be responsible
for preservation of the stereochemistry during alkene
epoxidation.
Albeit there is agreement with r espect to the occurrence of
cationic rearrangement products in P450-catalyzed reac-
tions, the mec hanism of t heir formation s till remains a
matter of considerable debate. While investigations con-
ducted with enantiomerically enriched isotopomers of
1-alkyl-2-arylcyclopropane and CYP2 members claimed t o
have disclosed d iscrepancies between the experimental data
and values computed according t o t he TSR c oncept [ 99],
giving support to the OH
+
insertion theory in carbocation
generation, more recent DFT studies applying cyclopropane
derivatives have s hown a coherent match between the TSR
paradigm and experimental results, excluding the partici-
0.0
13.9
14.9
17.4
0.6

0.9
2.9
10.4
-25.5
-27.9
4,2
1 + C
2
H
4
4
TS1-IV
4
TS1
4
TS1-III
4
TS2-IV
4
TS2-III
2
3
4
3
4
2-IV
2
2-IV
4
2-III

4
2
cat
(xy)
4
2
cat
(z
2
)
2
2
cat
3.2
4
TS1-IV (
2
TS1-IV) {
4
TS1-III}
∠OCC: 90.5 {85.0}
4
TS2-IV {
4
TS2-III}
1.486
(1.482)
{1.488}
[1.465]
1.464

(1.473)
{1.469}
[1.395]
1.810
(1.808)
{1.821}
[1.978]
2.381
(2.421)
{2.447}
[2.362]
1.463 {1.485}
1.462 {1.499}
1.944 {1.882}
2.465 {2.523}
2.412 (2.501) {2.413}
1.743 (1.716) {1.750}
1.897 (2.039) {1.898}
1.396 (1.371) {1.392}
∠OCC: 107.0 (108.2) {107.7} [119.5]
∠FeOCC: -178.8 (-179.2) {-171.0} [53.7]
4
2-IV
rad
(
2
2-IV
rad
) {
4

2-III
rad
} [
2
2
cat
]
Scheme 10. Potential energy s urface for e thene
epoxidation. The energies for the low- and
high-spin states (
2,4
TS1), the corresponding
transition intermediates (
2,4
2) and epoxide
products (
2,4
3) are expressed in kcalÆmol
)1
.
Data taken from [327] with permission.
4350 P. Hlavica (Eur. J. Biochem. 271) Ó FEBS 2004
pation of free carbocation d uring C-H hydroxylation of t he
alkanes [328]. H ence, cationic i ntermediates that appear as
intimate ion pair species [HSFe(III)porOH
–
/Alk
+
]have
been reasoned to arise either from minor dissociation of the

high-spin ion pairs or electron transfer from the corres-
ponding radical complexes [HSFe(IV)porOH/Alk
•
]. These
puzzling inconsistencies m ay be accomodated by assuming
that the Ôtwo-oxidantÕ and Ôtwo-stateÕ hypotheses are not
mutually exclusive (Scheme 2).
While there i s little d issent regarding the role of peroxo-
iron as an oxidant i n P 450-dependent nucleophilic reactions,
the interesting concept of Fe(III)-OOH or [Fe(III)-H
2
O
2
]
acting as alternative electrophilic oxygenating intermediates
needs more d irect evidence vis-a
`
-vis the TSR the ory . On the
other h and, intractability of h igh-energy intermediates such
as ferryl precludes facile experimental proof of the Ôtwo-
stateÕ proposal owing to l ack of a ppropriate technical
means. Hence, new perspectives for increasing the siz e of the
computational models, such as the combined quantum
mechanical/molecular mechanical (QM/MM) meth ods
[329], may afford truly innovative solutions for differenti-
ating the complex pathways o f P450-catalyzed oxygen
activation on a theoretical basis.
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