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-

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: [email protected]

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

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)

(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

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

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

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

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

[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

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

)
] 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

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

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

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

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

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

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-

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

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

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-

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-

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

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

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

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.

)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)

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

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

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

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

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

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

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

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

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

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

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

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

(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

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.
References
1. Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J.,
Feyereisen, R., Waxman, D.J., Waterman, M.R., Gotoh, O.,

tion reactions. Acc. Chem. Res. 33, 449–455.
10. Newcomb, M., H ollenberg, P.F. & Coon, M.J. (2003) Multiple
mechanisms and multiple oxidants in P450-catalyzed hydro-
xylations. Arch. Biochem. Biophys. 409, 7 2–79.
11. Vaz, A.D.N., Roberts, E.S. & Coon, M.J. (1991) Olefin
formation i n the oxidative deformylation of aldehydes by
cytochrome P450. Mechanistic implications for catalysis by
oxygen-derived peroxide. J. Am. Chem. So c. 113, 5 886–5887.
12. Coon,M.J.,Vaz,A.D.N.,McGinnity,D.F.&Peng,H.M.(1998)
Multiple activated oxygen species in P450 catalysis. Contribu-
tions to s pec ificity in drug m etabo lism. Drug Metab. Dispos. 26,
1190–1193.
13. Vaz, A.D.N. (2001) Multiple oxidants in cytochrome P450-
catalyzed reactions: implications for drug metabolism. Curr.
Drug Me tab. 2, 1 –16.
14. Watanabe, Y. (2001) Alternatives to the oxoferryl p orphyrin
cation radical as the pr oposed reac tive intermediate o f cyto-
chrome P450: two -electron oxidized Fe(III) porphyrin deriva-
tives. J. Biol. Inorg. C hem. 6, 8 46–856.
15. Coon, M.J. (2003) Multiple oxidants and m ultiple m echanisms i n
cytochrome P450 catalysis. Biochem. Biophys. Res. Commun.
312, 1 63–168.
16. Ortiz d e Montellano, P.R. & Stearns, R.A. (1987) Timing of the
radical recombination step in cytochrome P450 catalysis with
ring-stained pr obes. J. Am. Chem. Soc . 109, 3 415–3420.
17. Atkinson, J.K. & In gold, K .U. ( 1993) Cytochrome P450
hydroxylation of hydrocarbons: variation in the r ate o f oxygen
rebound u sin g cyclopropyl radical clo ck s in clud ing two new ul-
trafast p robes. Biochemistry 32, 9209–9214.
18. Oprian, D.D., G orsky, L.D. & Coon, M .J. ( 1983) Properties of

nated form of the hemopro tein. Arch. Biochem. Biophys. 149,
197–208.
26. Eisenstein, L., D ebey, P. & Douzou, P. (1977) P450cam: oxy-
genated c omplexes stabiliz ed at low temperature. Bioc hem. Bio-
phys. R es. Commun. 77, 1377–1383.
27. Kerekjarto, B. & Staudinger, H. (1966) Die Sauer-
stoffabha
¨
ngigkeit der HydroxylierungvonAcetanilidzu
4-Acetaminophe nol du rch Kaninchenleb ermikro somen. Hoppe-
Seyler Z. Physiol. Chem . 347, 7 –17.
28. Hlavica, P. (1971) Hepatic mixed function amine oxidase. An
allosteric system. Xenobiotica 1, 537–538.
29. Hlavica, P. (1972) Interaction of oxygen and aromatic amines
with hepatic m icrosomal m ixe d-function o xid ase. Biochim. Bio-
phys. A cta 273, 318– 327.
30. Hlavica, P. & Kehl, M. (1974) Studies on the mechanism o f
hepatic microsomal N-oxide formation. I. Effect of carbon
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4351
monoxide on t he N-oxida tion of N,N-dimethylaniline. Hoppe-
Seyler Z. Physiol. Chem . 355, 1 508–1514.
31. Gasser, R., Negishi, M. & Philpot, R.M. (19 88) Primary s truc-
tures of m ultiple f orms of cytochrome P-450 isozyme 2 derived
from rabbit p ulmon ary and hepatic cDNAs. Mol. Pharmacol. 32,
22–30.
32. Hlavica, P. & Lewis, D.F.V. (2001) Allosteric phenomena in
cytochrome P450-catalyzed monooxygenations. Eur. J. Biochem.
268, 4 817–4832.
33. Golly, I., H lavica, P. & Schartau, W. ( 1988) The functional r ole
of cytochrome b

M.D., Reid, G.A., Chapman, S.K. & Daff, S. (2003) Oxygen
activation and electron transfer i n flavocytochrome P 450 BM3.
J. Am. Chem. Soc. 125, 150 10–15020.
41. Schulze, J., Tscho
¨
p, K., Lehnerer, M. & Hlavica, P. (2000)
Residue 285 in cytochrome P450 2B4 lacking the NH
2
-terminal
hydrophobic sequence has a role in the functional association of
NADPH-cytochrome P45 0 redu ctase. Biochem. Biophys. Res.
Commun. 270, 777–781.
42. Bonfils, C., Balny, C., Douzou, P. & Maurel, P. (1980) Stud ies on
the reactivity of the oxyferro intermediate of highly purified mi-
crosomal P-450. In Biochemistry, B iophysics and Regulation of
Cytochrome P-450 (Gu stafsson, J.A., Carlstedt-Duke, J., Mode,
A. & Rafter, J., eds), pp. 559–564. Elsevier, Am sterdam.
43. Lipscomb, J .D., Sligar, S.G., Namtvedt, M .J. & Gunsalus,
I.C. (1976) Autooxidation and hydroxylation reactions of
oxygenated cytochrome P-450cam. J. Biol. Chem. 251, 1116–
1124.
44. Kuthan, H., Tsuji, H., Graf, H., Ullrich, V., Werringloer, J . &
Estabrook, R.W. (1978) Generation o f superoxide anion as a
source of hydrogen peroxide in a reconstituted monooxygenase
system. FEBS Lett. 91 , 343–345.
45. Loew,G.H.&Harris,D.L.(2000)Roleofthehemeactivesite
and protein environm ent in s tructure, spectra, an d function o f
the cytochrome P450s. Chem. Rev. 100, 407–419.
46. Aikens, J. & Sligar, S .G. (1994) Kinetic solvent isotope effects
during oxygen activation by cytochrome P-450cam. J. Am.

reaction intermediates o f cytochrome P450 studied by
radiolytic reduction with phosphorus-32. J. Biol. Chem. 276,
11648–11652.
55. Makris, T.M., Davydov, R., Denisov, I.G., Hoffman, B.M. &
Sligar, S.G. (2002) Mechanistic enzymology of oxygen activation
by the cytoc hromes P450. Dr ug Metab. Rev. 34, 691–708.
56. Denisov, I.G., Hung, S.C., Weiss, K.E., Mc Lean, M .A., Shiro,
Y., Park, S.Y., Champion, P.M. & Sligar, S.G. (2001) Char-
acterization of the oxygenated intermediate of the thermophilic
cytochrome P450 CYP119. J. Inorg. Biochem. 87, 215–226.
57. Kobayashi, K., Iwamoto, T. & Honda, K. (1994) Spectral
intermediate in the reaction of ferrous cytochrome P450cam with
superoxide anion. Biochem. Biophys. Res. Commun. 201, 1348–
1355.
58. Benson, D.E., Suslick, K.S. & Sligar, S.G. (1997) Reduced o xy
intermediate observed in D251N cytochrome P450cam. Bio-
chemistry 36, 5104–5107.
59. Kamachi, T. & Yoshizawa, K. (2003) A theoretical study o n the
mechanism o f cam phor h ydroxylation by compound I of cyt o-
chrome P450. J. Am. Chem. Soc. 125, 4652–4661.
60. White, R.E. & Coon, M.J. (1980) Oxygen activation by cyto-
chrome P-45 0. Annu. R ev. Biochem. 49 , 315–356.
61. Macdonald, T .L., Gutheim, W.G., Martin, R.B. & Guengerich,
F.P. (1989) Oxidation of substituted N,N-dimethylanilines by
cytochrome P-450: estimation of the effective oxidation-reduc-
tion potential of cytochrome P-450. Biochemistry 28, 2071–2077.
62. Sono, M., And ersson, L .A. & Dawson, J.H. ( 1982) S ulfur don or
ligand binding to ferric cytochrome P450cam and myo globin.
Ultraviolet-visible absorption, magnetic c ircular d ichroism, and
electron paramagnetic resonance spectrosocopic investigation of

function of liver microsomal cytochrome P-450: comparison of
hydrogen peroxide and NADPH-catalysed N-demethylation re-
actions. Xenobiotica 14, 87–104.
72. Blake, R.C. & Coon, M.J. (1989) On the mechanism of action
of cytochrome P-45 0: spectral in termediates in t he reaction with
iodosobenzene an d its de rivatives. J. Biol. Chem . 264, 3694–
3701.
73. Rahimtula, A.D., O’Brien, P.J., H rycay, E.G ., Peterson, J .A. &
Estabrook, R.W. ( 1974) Possible higher valence states of c yto-
chrome P-450 d uring oxidative reactions. Biochem. Biophys. Res.
Commun. 60, 695–702.
74. Gustafsson, J.A., Hrycay, E.G. & Ernster, L. (1976) S odium
periodate, sodium chlorite, and organic hydroperoxides as
hydroxylating agents in s teroid hydroxylation reactions catalyzed
by adrenocortical microsomal an d mitocho ndrial cytochrome P -
450. Ar ch. Biochem. B iophys. 174, 4 40–453.
75. Egawa, T., Shimada, H. & Ishim ura, Y. (1994) Evidence for
compound I formation in the reaction of cytochrome P450cam
with m-chloroperbenzoic acid. Biochem. Bio phys. Res. Commu n.
201, 1464–1469.
76. de Visser, S.P., Shaik, S., Sharma, P.K., Kumar, D. & Thiel, W.
(2003) Active species of horseradish peroxidase (HRP) and
cytochrome P450: t wo e lectronic c hame leons. J. Am. Chem. Soc.
125, 15779–15788.
77. Mansuy, D., Battioni, P. & Battioni, J.P. (1989) Chemical model
systems for drug-metabolizing cytochrome-P-450-dependent
monooxygenases. Eur. J. Biochem. 184 , 267–285.
78. Groves, J.T., Haushalter, R.C., Nakamura, M., Nemo, T.E. &
Evans, B.J. (1 981) High-valent i ron-porphyrin complexes related
to peroxidase and cytochrome P-450. J. Am. Chem. Soc. 103,

86. Guallar,V.,Baik,M.H.,Lippard,S.J.&Friesner,R.A.(2003)
Peripheral heme substitue nts control the hydrogen-atom
abstraction c hemistry in cytochromes P450. Proc. Natl Acad. Sci.
USA 100, 6998–7002.
87. Groves, J.T., McClusky, G.A., White, R.E. & Coon, M.J. (1978)
Aliphatic hydroxylation by highly purified liver microsomal
cytochrome P-450. Evidence for a carbon radical i ntermediate.
Biochem. Biop hys. Res. Commun. 81, 154–160.
88. Gelb, M.H., Heimbrook, D.C., Ma
¨
lko
¨
nen, P. & Sligar, S.G.
(1982) St ereoch emistry and deute rium i sot ope effects in camphor
hydroxylation by the cytochrome P4 50cam m onooxygenase
system. Bioc hemistry 21, 370–377.
89. Groves, J.T. & Subramanian, D .V. (1984) Hydroxylation by
cytochrome P -450 and metalloporphyrin models. Evidence for
allylic rearr angements. J. Am . Chem. Soc. 106, 2177–2181.
90. Frommer, U., Ullrich, V. & Staudinger, H.J. (1970) Hydro-
xylation of aliphatic compounds by liver microsomes, I. The
distribution pattern of isomeric alcoho ls. Hoppe-Seyler Z. Phy-
siol. C hem. 351, 903 –912.
91. Hjelmeland, L.M., Aronow, L. & Trudell, J.R. (1977)
Intramolecular determination of primary kinetic isotope effects
in hydroxylations catalyzed by cytochrome P-450. Biochem.
Biophys. R es. Commun. 76, 541–549.
92. Kadkhodayan, S., Coulter, E.D., Maryniak, D.M., Bryson,
T.A. & Dawson, J.H. (1995) Uncoupling oxygen transfer and
electron transfer in the oxygenatio n o f camp hor analogues by

101. Volz, T.J., Rock, D .A. & Jones, J.P. (2002) Evidence for two
different active oxygen species in cytochrome P450BM 3 medi-
ated sulfoxidation and N-dealkylation r eactions. J. Am. Chem.
Soc. 124, 9724–9725.
102. Matsunaga, I., Yamada, A ., Lee, D .S., O bayashi, E., Fujiwara,
N., Kobayashi, K. , Ogura, H . & Shiro, Y. (2002) Enzymatic
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4353
reaction of hydrogen peroxide-dependent peroxygenase
cytochrome P450s: kinetic deuterium isotope effects and ana-
lyses by r esonance Raman spectroscopy. Biochemist ry 41, 1886–
1892.
103. Newcomb, M ., Shen, R., Lu, Y., C oo n, M.J., H ollenbe rg, P.F.,
Kopp, D .A. & Lippard, S.J. ( 2002) E valu ation of n orcarane as a
probe f or radicals in cytochrome P450- and soluble methane
monooxygenase-catalyzed hydroxylation reactions. J. Am.
Chem. Soc. 124, 6879–6886.
104. Newcomb, M., Le T adic, M .H., Put t, D. A. & Hollenberg, P.F.
(1995) An incre dibly fast apparent oxygen re bound r ate c onstant
for hydrocarbon hydroxylation by c ytochrome P -450 enzymes.
J. Am. Chem. Soc. 117, 331 2–3313.
105. Toy,P.H.,Newcomb,M.,Coon,M.J.&Vaz,A.D.N.(1998)
Two distinct e lectrophilic oxidants effect hydroxylation in
cytochrome P-450-catalyzed reactions. J. Am. C hem. Soc. 120,
9718–9719.
106. Newcomb, M., Le Tadic-Biadatti, M.H., Chestney, D.L.,
Roberts, E.S. & Hollenberg, P.F. (1995) A nonsynchronous
concerted mechanism for cytochrome P-450 catalyzed hydro-
xylations. J. Am. C hem. Soc. 11 7, 12085–12091.
107. Newcomb, M., Shen, R., Choi, S.Y., Toy, P .H., Hollenberg,
P.F., Vaz, A.D.N. & Coon, M.J. (2000) Cytochrome P450-cat-

simulations of the resting and hy drogen peroxide-bound states of
cytochrome c peroxidase. Bio chemistry 31, 1 1166–11174.
113. Harris, D.L. & Loew, G.H. (1996) Identification of putative
peroxide intermediates of peroxidases by electronic structure and
spectra calculations. J. Am. Chem. Soc . 118, 10588–10594.
114. Dawson, J .H. (1988) P robing structure-function relationships i n
heme-containing o xygenases a nd peroxidases. Science 240, 433–
439.
115. Hata, M., Ho shino, T. & Tsuda, M. (2000) An ultimate species in
the substrate oxidation process by cytochrome P-450. Chem.
Commun. 2037–2038.
116. Hlavica, P., Golly, I. & Mietaschk, J. (1983) Compara tive studies
on the cumene hydroperoxide- and NADPH-supported
N-oxidation of 4-chloroaniline by cytochrome P-450. Biochem. J.
212, 5 39–547.
117. Hlavica, P. (1982) Biological oxidation of nitrogen in organic
compounds and d isposition o f N-oxidized products. CRC Crit.
Rev. Biochem. 12, 39–101.
118. Zang, Y., Elgren, T.E., Dong, Y.L. & Que, Y. (1993) A high-
potential ferrous complex and it s conversion to an alkylperoxo-
iron(III) intermediate. A lipoxygenase model. J. Am . C hem. So c.
115, 8 11–813.
119. Wada, A., Ogo, S., Watanabe, Y., Mukai, M., Kitagawa, T.,
Jitsukawa, K., Masuda, H. & Einaga, H. (1999) Synthesis and
characterization of novel alkylperoxo m ononuclear i ron(III)
complexes with a tripodal pyridylamine ligand: a model for
peroxo intermediates in reactions catalyze d by non-heme iro n
enzymes. Inorg. Chem. 38, 3592–3593.
120. Balch, A.L. (199 2) The reactivity of spectroscopically detected
peroxy complexes of i ron porphyrins. Inorg. Chim. A cta 198–200,

mechanism. Drug Metab. Rev. 30, 739–786.
129. McCandlish, E., Miksztal, A.R., N appa, M., Sprenger, A .Q.,
Valentine, J.S., Stong, J.D. & Spiro, T.G. (1980) Reactions of
superoxide wit h iron porphyrins i n aprotic solvents. A high spin
ferric p orphyrin peroxo c omplex. J. Am. Chem. Soc. 102, 4268–
4271.
130. Welborn, C.H., D olphin, D. & James, B.R. (1981 ) One-electron
electrochemical reduction of a ferrous po rp hyrin dioxygen
complex . J. Am. Chem. Soc . 103, 2 869–2871.
131. Fridovich, I. (1986) Biological effects of the superoxide radical.
Arch. Biochem. B ioph ys. 247, 1 –11.
132. Sauer-Masarwa, A., Herron, N., Fendrick, C.M. & Busch, D.H.
(1993) Kinetics and intermediates in the autoxidation of syn-
thetic, non-porphyrin iron(II) dioxygen c arriers. Inorg. Chem. 32,
1086–1094.
133. Sutton, H.C., R oberts, P.B. & Winterbourn, C.C. (1976) The r ate
of reaction of su peroxide radical ion wit h oxyhaemoglobin and
methaemoglobin. Bioc hem. J. 155, 503–510.
134. Kobayashi, K. & Hayashi, K. (1981) One-electron
reduction in oxyform of hemoproteins. J. Biol. Chem. 256,
12350–12354.
135. Peterson, J.A. & Graham-Lorence, S.E. (19 95) Ba cterial P450s.
Structural similarities and functional differences. In Cytochrome
P450: Structure, Mechanism, and Biochemistry (Ortiz de Mon-
tallano, P.R., ed.), 2nd edn, pp. 151–180. Plenum Press, New
York.
136. Martinis, S .A., Atkins, W.M., Stayton, P.S. & Sligar, S .G. (1989)
A conserved residue o f cytochrome P-450 is involved in heme-
oxygen stability and activation. J. Am. Ch em. Soc. 111 , 9252–
9253.

B.W. (1957) Erythrocyte metabolism. IV. I solation and proper-
ties of m ethemoglo bin reductase. J. Bio l. Chem. 227, 261–272.
145. Passon, P.G. & Hultquist, D.E. (1972) Soluble cytochrome b
5
reductase from human erythrocytes. Biochim. Biophys. Acta 275,
62–73.
146. Starke, D.W. & Mieyal, J.J. (1989) Hemoglobin catalysis of a
monooxygenase-like aliphatic hydroxylation reaction. Biochem.
Pharmacol. 38, 201–204.
147. Starke, K.S., Blisard, D.W. & Mieyal, J .J. (1984) S ubstrate spe-
cificity of the monooxygenase activity of hemoglobin. Mol.
Pharmacol. 25, 467–475.
148. Blisard, K.S. & M ieyal, J.J. (1979) Characterization of the aniline
hydroxylase a ctivity of e rythrocytes. J. Biol. Chem. 254, 5104–
5110.
149.Mieyal,J.J.,Ackerman,R.S.,Blumer,J.L.&Freeman,L.S.
(1976) Characterization of enzyme-like activity of human
hemoglobin. Properties o f the hemoglobin-P-450 reductase–
coupled aniline h ydroxylase s ystem. J. Biol. Chem. 251, 3436–
3441.
150. Brown, W.D. & S nyder, H.E. (1969) Nonenzymatic reduction
and oxidatio n of m yoglobin and hemoglobin by n icotinamide
adenine dinucleot ides and flavins. J. Bi ol. Chem. 244, 6 702–6706.
151. Adams, P.A. & Berman, M.C. (1982) Hemin-mediated para
hydroxylation of aniline: a potential model for oxygen activation
and insertion reactions o f mixed function oxidases. J. Inorg.
Biochem. 17 , 1–14.
152. Mieyal, J.J. & Blume r, J.L. ( 1976) A cceleration of t he auto-
oxidation o f human oxyhemoglobin b y aniline and its relation to
hemoglobin-cat alyzed aniline hyroxyla tion. J. Biol. Chem. 251,

157. Gasyna, Z. (1979) Intermediate spin-states in one-electron
reduction o f oxygen-hemoprotein c omp lexes at low t empe rature.
FEBS Lett. 106, 213–218.
158. Ibrahim, M., Denisov, I.G., Makris, T.M., Kincaid, J.R. & S li-
gar, S.G. (2003) Resonance Raman spectroscopic s tudies of hy-
droperoxo-myoglobin at c ryogenic temperatures. J. Am. C hem.
Soc. 125, 13714–13718.
159. Egawa, T., Yoshioka, S., Takahashi, S., Hori, H., Nagano, S.,
Shimada, H ., Ishimori, K., Morishima, I ., Suematsu, M . & Ish-
imura, Y. (200 3) Kinetic and spectroscopic c haracterization of a
hydroperoxy compound i n the react ion of native myoglobin w it h
hydrogen peroxide. J. Biol. C hem. 278, 4 1597–41606.
160. Alvarez, J.C. & Ortiz de Montellano, P.R. (1992) Thianthrene
5-oxide as probe of the electrophilicity of hemoprotein oxidizing
species. Bioc hemistry 31, 8315–8322.
161. Miksztal, A.R. & Valentine, J.S. (1984) Reactivity of t he peroxo
ligand in metalloporphyrin complexes. Reaction o f sulfur dioxide
with iron and titanium p orphyrin peroxo comple xes to g ive sul-
fato complexes or s ulfate. Inorg. Chem . 23, 3548–3552.
162. Lenk, W. & Sterzl, H. (1984) Peroxidase activity of oxyhe-
moglobin in vitro. Xenobiotica 14 , 3548–3552.
163. Golly, I. & Hlavica, P. (1983) The role of hemoglobin in the
N-oxidation of 4-chloroaniline. Biochim. Biophys. Acta 760,69–
76.
164. Golly, I. & Hlavica, P. (1983) Mechanisms of extrahepatic
bioactivation of aromatic amines: the r ole of he moglobin in the
N-oxidation of 4-chloraniline. In Extr ahepatic Drug Me tabolism
and Chemical Carcinogenesis (Rydstro
¨
m, J., Montelius, J. &

evidence for an electrophilic, f erric peroxide species. Acc. Chem.
Res. 31, 543–549.
170. Ortiz d e M ontellano, P.R. & Wilks , A. (2001) H eme oxy genase
structure and mechanism. Adv. Inorg. Chem. 51, 359–407.
171. Lad,L.,Schuller,D.J.,Shimizu, H., Friedman, J., Li, H., Ortiz
de Montellano, P.R. & Poulos, T.L. (2003) Comparison of the
heme-free and -bound c rystal structures of human heme oxyge-
nase-1. J. Bio l. Chem. 278, 7834–7843.
172. Beale, S.I. (1993) Biosynthesis of phyc obilins. Chem. R ev. 93,
785–802.
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4355
173. Wilks, A. & Schmitt, M.P. ( 1998) E xpression and ch aracteriza-
tion of hem e oxyge nase (H mu O ) from Corynebacterium diph-
theriae. J. Biol. C hem. 273, 837–841.
174. Wilks, A. & Ortiz de Montellano, P.R. (1993) Rat live r heme
oxygenase. High level expressionofatruncatedsolubleformand
nature of the m eso-hydro xylating species. J. Biol. Chem. 268,
22357–22362.
175. Wilks, A., Torpey, J. & Ortiz de Montellano, P.R. ( 1994) Heme
oxygenase (HO-1). Evidence for electrophilic oxygen addition to
theporphyrinringintheformationofa-meso-hydroxyheme.
J. Bi ol. Chem. 269 , 29553–29556.
176. Torpey, J . & Or tiz de M ontellano, P.R. ( 1996) Oxidation of the
meso-methylmesoh eme regioisomers by heme oxyg enase. Elec-
tronic control of t he reaction regiosp ecificity. J. Biol. Chem. 271,
26067–26073.
177. Torpey, J. & Ortiz d e M ontellano, P.R. (1997 ) O xidation of a-
meso-formylmesohem e by heme oxyge nase. Electronic control o f
the r eaction regiospecificity. J. Biol. Chem. 272, 22008–22014.
178. Avila,L.,Huang,H.,Damaso,C.O.,Lu,S.,Moenne-Loccoz,P.

octapeptide (microperoxidase-8) and identification of the
monomeric form in a queous solution. J. I norg. Biochem. 27, 227–
243.
184. Osman,A.M.,Koerts,J.,Boersma,M.G.,Boeren,S.,Veeger,C.
& Rietjens, I.M.C.M. (1996) Microperoxidase/H
2
O
2
-catalyzed
aromatic hydroxylation proceeds by a cytochrome-P-450-type
oxygen-transfer me chanism. Eur. J. Biochem. 240, 2 32–238.
185. Boersma, M.G., Primus, J.L., Koerts, J., Veeger, C. & Rietjens,
I.M.C.M. (2000) H eme-(hydro) peroxide mediated O-and
N-dealkylation . A study with microperoxidase. Eur. J. Bioc hem.
267, 6 673–6678.
186. Primus, J.L., B oersma, M.G., Mandon, D., B oeren, S., V eeger,
C., W eiss, R . & Rietjens, I.M.C.M. (1999) The effect of iron to
manganese substitution on microperoxidase 8 catalysed per-
oxidase and cytochrome P450 type of catalysis. J. Biol. I norg.
Chem. 4, 274–283.
187. Rusvai, E., Vegh, M., Kramer, M. & Horvath, I. (1988)
Hydroxylation o f aniline mediated by heme-bound oxy-radicals
in a heme peptide model system. Biochem. Pharmacol. 37, 4574 –
4577.
188. van Haandel, M.J.H., Primus, J.L., T eunis, C., B oersma, M.G.,
Osman, A.M., Veeger, C. & Rietjens, I.M.C.M. (1998)
Reversible for mation of high-valent-iron-oxo porphyrin in ter-
mediates in h eme-based catalysis: re visiting the kinetic model for
horseradish p eroxidase. Inorg. Chim. A cta 275–276, 98–105.
189. Dorovska-Taran, V., Posthumus, M.A., Boeren, S., B oersma,

lating dehalogenation of halophenol derivatives in alcoholic
media. Proc. N atl Acad. Sci. USA 94 , 4295–4299.
194. Wang, J.S., Baek, H.K. & van Wart, H.E. (1991) High-valent
intermediates in the reaction of N-acetyl microperoxidase-8 with
hydrogen peroxide: models for c om pounds 0, I and II o f horse-
radish peroxidase. Biochem. Biophys. Res. Commun. 179, 1320–
1324.
195. Primus, J .L., Grunenwald, S., Hagedoorn, P.L., Albrecht-Gray,
A.M., Mandon, D. & Veeger, C. (2002) The nature of the
intermediates in the react ions of Fe(III)- and Mn(III)-micro-
peroxidase-8 with H
2
O
2
: a rapid kinetics study. J. Am. C hem.
Soc. 124, 1 214–1221.
196. Machii, K., W atanabe, Y. & Morishima, I. (1995) Ac ylperoxo-
iron(III) porphy rin complexes: a new entry of potent oxidants for
the alkene ep oxidation. J. Am. Chem. Soc . 117, 6 691–6697.
197. Suzuki, N., Higuchi, T. & Nagano, T. (2002) Multiple ac tive
intermediates in oxidation reaction catalyzedbysyntheticheme-
thiolate complex r elevant to c ytochrome P450. J. Am. Chem. Soc.
124, 9 622–9628.
198. Nam, W., Lim, M.H., Moon, S .K. & Kim, C. (2000) Participa-
tion of two distinct hydroxylating intermediates in iron(III)
porphyrin complex-catalyzed hydroxylation of alkanes. J. Am.
Chem. Soc. 122, 10805–10809.
199. Fontecave, M. & Mansuy, D. (1984) Monooxygenase-like o xi-
dations of olefins and alkanes catalyzed by manganese por-
phyrins: comparison of systems involving either O

205. Batie, C.J., LaHaie, E. & Ballou, D.P. (1987) Purification and
characterization of phthalate oxygenase and phthalate oxygenase
reductase f rom Pseu domonas c epacia. J. Biol. Chem. 262, 1510–
1518.
206. Wackett, L.P., Kwart, L.D. & Gibson, D.T. (1988) Benzylic
monooxygenation catalyzed by toluene d ioxygenase from Pseu-
domonas putida. Biochemistry 27, 1360–1367.
207. Resnick, S.M., Lee, K. & Gibson, D .T. (1996) Diverse reactions
catalyzed by naphthalene dioxygenase from Pseudomonas sp.
strain NCI B 9816. J. Ind. M icrobiol. 17 , 438–457.
208. Kauppi, B., L ee, K., Carredano, E ., Parales, R.E., G ibson, D.T.,
Eklund, H. & Ram aswamy, S. (1998) S tructure of an aromatic
ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase.
Structure 6, 571–586.
209. Que, L. (2000) One motif – many d ifferent r eactions. Na t. Struct.
Biol. 7, 182–184.
210. Karlsson, A., Parales, J.V., Parales, R.E., Gibson, D.T., Eklund,
H. & Ramaswamy, S. (2003) Crystal structure of naphthalene
dioxygenase: s ide-on binding of dioxygen t o iron. Science 299,
1039–1042.
211.Wolfe,M.D.,Parales,J.V.,Gibson,D.T.&Lipscomb,J.D.
(2001) Single turnover chem istry and regulation of O
2
activation
by the oxygenase c omponen t of naphthalene 1,2-dioxygenase.
J. Biol. C he m. 276, 1945–1953.
212. Wolfe, M.D. & Lipscomb, J.D. (2003) Hydrogen peroxide-cou-
pled cis-diol formation c atalyzed b y naphthalene 1,2-dioxygen-
ase. J. Biol. Chem. 278, 829–835.
213. Lee, K. (1999) Benzene-induced uncoupling of naphthalene

nck, E.
& Lipscomb, J.D. (2002) Benzoate 1,2-dioxygenase from
Pseudomonas putida: single turnover kinetics and regulation of a
two-component Rieske dioxygenase. Biochemistry 41, 9611–
9626.
222. Ballou, D. & B atie, C. ( 1988) Phthalate oxygenase, a Rieske iron-
sulfur protein from Pseudomonas cepacia.InOxidases
and Related Redox Systems (King,T.E.,Mason,H.S.&
Morrison, M., e ds), pp. 211–226. A lan R. Liss, Inc., N ew York.
223. Solomon, E.I., Brunold, T .C., Dav is, M.I., Kemsley, J .N., Lee,
S.K., Lehnert, N., Nee se, F., Skulan, A.J., Yang, Y.S . & Zhou, J.
(2000) Geometric and electronic structure/ function corre latio ns
in non-heme iron e nzymes. Chem. Rev. 100, 2 35–349.
224. Chen,K.,Costas,M.,Kim,J.,Tipton,A.K.&Que,L.(2002)
Olefin cis-dihydroxylation versus e p oxidation by n on- heme iron
catalysts: two faces of an Fe(III)-OOH coin. J. Am. C hem. Soc.
124, 3 026–3035.
225. Chen, K., Costas, M. & Que, L. (2002) Spin state t uning of non-
heme iron-catalyzed h ydrocarbon oxidations: participation of
Fe(III)-OOH an d F e(V) ¼O intermediates. J. Chem. S o c., Dalton
Trans. 67 2–679.
226. Hecht, S.M. (2000) Bleomycin new perspectives on the mech-
anism o f action. J. Nat. Prod. 63, 158–168.
227. Burger, R .M. (1998) Cleavage of nucleic acids by bleomycin.
Chem. Rev. 98 , 1153–1169.
228. Loeb, K .E., Zaleski, J.M., Westre, T.E., Guajardo, R.J., Mas-
charak, P.K., Hedman, B., Ho dgson , K .O. & Solomon, E.I.
(1995) Sp ectrosco pic definition of the geometric and electronic
structure of t he non-heme iron active site in iron(II) bleomycin:
correlation with oxygen reactivity. J. Am. Chem. Soc. 117, 4545–

O o xygen,
14
N, and exchangeable
1
H. J. Am. C hem.
Soc. 117, 7508–7512.
236. Boger, D.L., Ramsey, T .M., Cai, H., Hoehn, S.T. & Stubbe, J.A.
(1998) A systematic evaluation of the bleomycin A2, 1-threonine
side chain: its role in preorganization of a compact conformation
implicated in sequence-selective DNA cleavage. J. Am. Chem.
Soc. 120, 9139–9148.
237. Ishida, R. & Takahashi, T. (1975) Incr eased DNA chain break-
age by com bined a ction of bleomycin and superoxide radic al.
Biochem. Biop hys. Res. Commun. 66, 1432–1438.
238. Ciriolo, M.R., Magliozzo, R.S. & Peisach, J. (1987) Microsome-
stimulated activation o f ferrous bleomycin in the presence of
DNA. J. Biol. Chem. 26 2 , 6290–6295.
239. Stubbe,J.A.,Kozarich,J.W.,Wu,W.&Vanderwall,D.E.(1996)
Bleomycins: a structural model for specificity, binding, and
double strand c leavage. Acc. Ch em. Res. 29, 3 22–330.
240. Burger, R.M. (2000) Nature of activated bleomycin. Struct.
Bond. 97 , 287–303.
241. Neese, F., Zaleski, J.M., Loeb-Zaleski, K. & Solomon, E.I.
(2000) Electronic structure of a ctivated bleomycin: oxygen
Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur. J. Biochem. 271) 4357
intermediates in heme v ersus non-heme iron. J. Am. Chem. Soc.
122, 1 1703–11724.
242. Wu, W., Vanderwall, D.E., Turner, C.J., Kozarich, J.W. &
Stubbe, J. (1996) Solution structure o f Co bleomycin A2 g reen
complexed with d(CCAGGCCTGG). J. Am. Chem. Soc. 118,

1565.
248. Bukowski, M.R., Z hu, S ., Koehntop, K.D., Brennessel, W.W. &
Que, L. (2004) Char acterization of an Fe
III
-OOH species and its
decomposition p roduct in a bleomycin model s ystem. J. Biol.
Inorg. Che m. 9, 39– 48.
249. Girerd, J.J., Banse, F. & Simaan, A.J. (2000) Characterization
and properties o f non-hemeironperoxocomplexes.Struct. Bond.
97, 1 45–177.
250. Neese, F. & Solomon, E.I. (1998) D etailed spectroscopic and
theoretical studies on [Fe(ED TA)(O
2
)]
3–
: electronic structure of
the side-on ferric-peroxide bond and its relevance to reactivity.
J. Am. Chem. Soc. 120, 128 29–12848.
251. Ho, R.Y.N., Roelfes, G., Hermant, R ., Hage, R ., Feringa, B.L.
& Que, L. (1999) Resonance Raman evidence for the inter-
conversion between an [Fe
III
-g
1
OOH]
2+
and [Fe
III
-g
2

mes d’hydroxylation des hydro-
carbures. Tetrahedron 31 , 777–784.
258. Davis, R., Durrant, J .L.A. & Khan, M.A. (1988) A study of the
mechanism of alkane h ydroxylation using the Fepy
4
-
PhNHNHPh-O
2
system. Polyhedron 7, 425–438.
259. Sheu, C . & Sawyer, D.T. (1990) Activation of dioxygen by bis[(2-
carboxy-6-carboxylato)pyridine]iron(II) for the b romination (via
BrCCl
3
) and monooxyge nation (via PhN HNHPh) of satu rate d
hydrocarbons: reaction mimic for the m ethane mo no oxygenase
proteins. J. Am . Chem. Soc. 112, 8212–8214.
260. Hage, J .P. & Sawyer, D.T. (1995) Iron(II)/reductant (DH
2
)-
induced a ctivation of d ioxygen for th e hydroxylation of aromatic
hydrocarbons and phenols: reaction mimic for tyrosine hy-
droxylase. J. Am. Chem. Soc . 117, 5 617–5621.
261. Sawyer, D.T., Sugimoto, H . & Calderwood, T.S. (1984) Base
(O
ÁÀ
2
,e
–
,orOH
–

267. Cole, P.A. & Robinson, C.H. ( 1990) Mechanism a nd inhibition
of cytochrome P-450 aromatase. J. M ed. Chem. 33, 2 933–2942.
268. Oh, S.S. & Robinson, C.H. (1993) Mechanism of human pla-
cental aromatase: a new active site model. J. Steroid B iochem .
Mol. Bi ol. 44, 389–397.
269. Akhtar,M.,Calder,M.R.,Corina,D.L.&Wright,J.N.(1982)
Mechanistic studie s on C-19 demethylation i n oestrogen bio-
synthesis. Bioc hem. J. 201, 569–580.
270. Stevenson, D.E., Wright, J .N. & Akhtar, M. (1988)
Mechanistic consideration of P-450 dependent enzymic reac-
tions: studies on oestriol biosynthesis. J. Chem. Soc. Perkin
Trans. I, 2043–2052.
271. Fischer, R.T., Trzaskos, J.M., Magolda, R.L., Ko, S .S., Brosz,
C.S. & Larsen, B. (1991) Lanosterol 14a-m ethyl demethylase.
Isolation and characterization of the third metabolically gener-
ated oxidative demethylation intermed iate. J. Biol. Chem. 266,
6124–6132.
272. Shyadehi, A .Z., Lamb, D.C., Kelly, S.L., Kelly, D.E., Schunck,
W.H., Wright, J.N., Corina, D. & Akhtar, M. (1996) The
mechanism o f the a cyl-carbon bond cleavage re action catalyzed
by recombinant sterol 14a-demethylase of Candida albicans.
(other names are: lanostero l 14 a-demethylase, P-450
14DM
,and
CYP51). J. Biol. Chem. 271 , 12445–12450.
273. Roberts, E.S., V az, A.D.N. & Coon, M .J. (1991) C atalysis by
cytochrome P-450 of an oxidative reaction in xenobiotic a lde -
hyde metabolism: de formylation with o lefin formation. Proc.
NatlAcad.Sci.USA88, 8963–8966.
274. Kuo, C.L., Raner, G .M., Vaz, A.D.N. & Coon, M.J. (1999)

ferent from cytochromes P450 catalyzing monooxygenations. In
Cytochrome P450: Structure, Mechanism, a nd Biochemistry
(Ortiz de Montellano, P .R., ed.), 2nd edn, pp. 537–574. Plenum
Press, New York.
282. Renaud, J.P., Bouche r, J.L.,Vadon,S.,Delaforge,M.&
Mansuy, D. (1993) Particular ability of liver P450s3A to catalyze
the o xidation of N
x
-hydroxyarginine to citrulline and nitrogen
oxides a nd occurrence in NO synthases of a sequence very similar
to the h eme- bindin g s equenc e i n P 450s. Biochem. Biophys. Re s.
Commun. 192, 5 3–60.
283. Bredt,D.S.,Hwang,P.M.,Glatt,C.E.,Lowenstein,C.,Reed,
R.R. & Snyder, S.H. (1991) Cloned and expressed nitric oxide
synthase structurally resembles cytochrome P-450 reductase.
Nature 351, 714–718.
284. Siddhanta, U., Presta, A., Fan, B., Wolan, D., R ousseau, D. &
Stuehr, D . ( 1998) D om ain swapping in in ducible nitric-oxide
synthase. Electron transfer occurs between flavin and heme
groups loc ated on adjacent subunits in the d imer. J. Biol. Chem.
273, 18950–18958.
285. Fischmann,T.O.,Hruza,A.,Niu,X.D.,Fossetta,J.D.,Lunn,
C.A., Dolphin, E., Progay, A.J., Reichert, P., Lundell, D.J.,
Narula, S .K. & Weber, P.C. (1999) Structural c haracterization of
nitric oxide synthase isoforms reveals striking active-site con-
servation. Nat. Struct. Biol. 6, 233–242.
286. Klatt,P.,Schmidt,K.,Lehner,D.,Glatter,O.,Ba
¨
chinger, H.P.
& Mayer, B. (1995) Structural analysis of porc ine brain nitric

Baillie, T.A. & Tang, W. (2002) Cytochrome P450 3A4-mediated
oxidative conversion of a cyano to an amide group in the
metabolism of pinacidil. Biochemistry 41, 2712–2718.
293. Moali, C., B oucher, J.L., Renodon-Corniere, A., S tuehr, D.J. &
Mansuy, D. ( 2001) Oxidation of N
G
-hydroxyarginine analogues
and vario us N-hydroxyguanidines by NO synthase II: key role of
tetrahydrobiopterin in the reaction mechanism and substrate
selectivity. Ch em . Res. T oxicol. 14, 202–210.
294. Marletta, M.A. (1993) Nitric oxide synthase structure and
mechanism. J. Biol. Chem. 268, 1 2231–12234.
295. Mansuy, D., Boucher, J.L. & Clement, B. (1995) On the
mechanism of nitric oxide formation upon cleavage of C¼N
(OH) bonds by NO-synthases and cytochrome P450. Biochimie
77, 6 61–667.
296. Korth, H.G., Sustmann, R., Thater, C., Butler, A.R. & Ingold,
K.U. (1994) On the mechanism of nitric oxide synthase-catalyzed
conversion o f N-hydroxy-
L
-arginine t o citrulline and nitric oxide.
J. Bi ol. Chem. 269 , 17776–17779.
297. Nishida,C.R.,Knudsen,G.,Straub,W.&OrtizdeMontellano,
P.R. (2002) Elec tron supply and catalytic oxidation of n itrogen
by cytochrome P450 and nitric oxide synthase. Drug Metab. Rev.
34, 4 79–501.
298. Adak, S., Wang, Q. & Stuehr, D.J. ( 2000) Arginine conversion to
nitric oxide by tetra-hydro biopterin-fre e neuronal nitric-o xide
synthase. I mplications fo r mechanism. J. Biol. Chem. 275 , 33554–
33561.