Whereas the long-standing consensus view of the P450 mechanism implicates a high-valent iron-oxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound
Trang 1R E V I E W A R T I C L E
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 hydrocarbons Whereas the long-standing consensus
view of the P450 mechanism implicates a high-valent
iron-oxene species as the predominant oxidant in the radicalar
hydrogen abstraction/oxygen rebound pathway, more
recent studies on isotope partitioning, product
rearrange-ments with radical clocks, 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 catalysis
in addition to the electrophilic oxenoid-iron entity; this may
contribute to the remarkable versatility of P450s in substrate
modification Precedent to this mechanistic concept is given
by studies with natural and synthetic P450 biomimics Whilethe concept of an alternative electrophilic oxidant necessi-tates C-H hydroxylation to be brought about by a cationicinsertion process, recent calculations employing densityfunctional theory favour a two-state reactivity scenario,implicating the usual ferryl-dependent oxygen reboundpathway to proceed via two spin states (doublet and quar-tet); state crossing is thought to be associated with either aninsertion or a radicalar mechanism Hence, challenge tofuture strategies should be to fold the disparate and some-times contradictory data into a harmonized overall picture.Keywords: (hydro)peroxo-iron; iron-oxene; O2-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], are 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 relatedcompounds has extensively captured the imagination ofbiochemists owing to the unfavourable thermodynamics ofthe dissociation event [3] Early views of such a mechanismfocused on an oxygen insertion pathway promoted by anelectrophilic, high-valent iron-oxo species (compound I) [4].This hypothesis was soon supplanted by the hydrogenabstraction/oxygen rebound concept implicating the exist-ence of radical intermediates, as developed on the basis ofthe well-known chemical properties of peroxidases andporphyrin model systems [5,6] The mechanistic details ofoxygen transfer have been addressed elsewhere [7,8].Mounting evidence provided during the past decadesuggests that hydroxylation reactions are more complexthan previously anticipated, and are not compatible withthe idea of a single reaction pathway The picture began tocloud when the application of ultrafast radical clocks
to time the oxygen-rebound step disclosed the amounts ofrearranged products not to correlate with the radicalrearrangement rate constants [9] Moreover, the use of aprobe that could distinguish between radical and cationicspecies hinted at the interference of cationic rearrangements,predicting the hydroxylation to occur via an insertionreaction in place of abstraction and recombination [9] Theformer process thus necessitated the insertion into a C-Hbond of the elements of OH+, implying that the ultimateelectrophilic oxidant was either hydroperoxo-iron or iron-complexed hydrogen peroxide [10] In addition, examina-tion of the oxidative deformylation of cyclic aldehydes as amodel 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 ToxikPharmak-ologie, Goethestr 33, D-80336 Mu¨nchen, Germany.
Fax: +49 89 218075701, Tel.: +49 89 218075706,
E-mail: hlavica@lrz.uni-muenchen.de
Abbreviations: TSR, two-state reactivity; KIE, kinetic isotope effects;
Hb, haemoglobin; Mb, myoglobin; HO, heme oxygenase; 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 oxidoreductase (EC 1.6.2.4); heme oxygenase (EC 1.14.99.3);
phthalate oxygenase reductase (EC 1.18.1); phthalate dioxygenase
(EC 1.14.12.7); toluene 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 29 July 2004, revised 27 September 2004,
accepted 28 September 2004)
Trang 2substrates 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 goal of the present perspective is to provide a critical
update of several aspects of the current state of biochemistry
relating to the apparently complex machinery of dioxygen
activation, which is considered to possibly implicate
mul-tiple oxygenating species in P450 catalysis Emphasis will be
put on the evaluation of comparative studies with non-P450
hemoproteins, nonheme metalloenzymes as well as
bio-mimetic model systems to discuss the multiple oxidant vs
the two-state reactivity theory
Iron-oxene acting as an electrophilic oxidant
in P450-catalyzed hydroxylations
The consensus mechanism for hydrocarbon hydroxylation
by P450 enzymes involves hydrogen atom abstraction from
the hydrocarbon by a high-valent iron-oxo species, best
described as an O¼ Fe(IV) porphyrin p-cation radical,
followed by homolytic substitution of the alkyl radical thus
formed in the so-called oxygen rebound step [5–8]
(Scheme 1) Using CYP2B isoforms as the catalysts, radical
collaps was demonstrated to occur at highly variable rates
exceeding those of the gross molecular motions of many
enzyme-bound substrates and depending on the
stereo-chemical specificities of the compounds to be acted upon
[16,17] Reduction of ferric P450 to the ferrous state sets the
stage for dioxygen binding, the event that commits the
hemoprotein to the step-by-step production of the activeoxidant (Scheme 2) Association of dioxygen with ferrousmicrosomal CYP1A2 [18], certain CYP2B isoforms [19–21],and CYP2C3 [18] to yield hexacoordinate low-spin com-plexes has been shown to be characterized by absorptionbands around 420 and 557 nm in the absolute spectra andbroad maxima at about 440 and 590 nm in the differencespectra Similar optical perturbations were also observedupon O2 binding to so-called class I P450s, comprisingmitochondrial and bacterial isozymes such as CYP11A1[22–24] and CYP101 [25,26], respectively The rapid initialstep in molecular oxygen activation by both class I and class
II P450s, as measured at varying temperatures, usuallyexhibits monophasic kinetic behaviour, with the second-order rate constants ranging from 0.58 to 8.41· 106M )1Æs)1[18,20,24,25] Interestingly, the presence of certain substratessuch as aromatic amines appears to favour homotropiccooperativity in dioxygen binding to P450s: using livermicrosomal samples from untreated rabbits, the O2satura-tion kinetics for acetanilide 4-hydroxylation have beenreported to bear sigmoidal character corresponding to a Hillinteraction coefficient, n, of 2.2 [27] Similar experimentswith N-alkyl arylamines gave concave upward double-reciprocal plots of velocity vs O2concentration, 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 ion tration and was most pronounced at physiological pH,whereas CO, acting as a positive effector, abolishedautoactivation at all pH values examined (Fig 1) [30] Inview of the well-known microheterogeneity of several rabbitliver P450s [31], the amine-induced cooperativity in O2
concen-complexation has been argued to involve the equilibriumbetween multiple, kinetically distinct protein conformations[32] Alternatively, the oligomeric nature of P450 [33] mightoffer the possibility of substrate–specific subunit inter-actions, as has been proposed for the fractional saturation
of hemoglobin by dioxygen [34]
Results from resonance Raman spectroscopy [35] andMo¨ssbauer studies [36] with microbial CYP101 indicate that
Scheme 1 Rebound mechanism for P450-catalyzed hydroxylations.
Reproduced from [6] with permission.
O O
Fe (III)
O
O
Fe(III)O O H
Fe(IV)O
HO OH
oxo-ion, low spin (inserts O)
Trang 3inter-the oxy intermediate of P450 most likely exists in inter-the
low-spin ferric-superoxide form, with the sixth 3d electron
largely transferred to O2 in an autoxidative process
(Scheme 2) Spontaneous autodecomposition of
oxy-cyto-chrome 2B4 to release ferric pigment and superoxide [37]
has been shown to occur in a biphasic [21,38] or even
triphasic [39] fashion, while monophasic first-order kinetics
were observed for autoxidation of substrate-bound
adreno-cortical CYP11A1 [23,24] and bacterial CYPs 101 and 102
[25,26,40], as measured above 0C or at subzero
temper-atures Abortive decay of oxygenated P450 is retarded 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] Moreover, the steady-state level of oxyferrous
P450 has been recognized to be governed by the hydrogen
ion concentration and ionic strength of the reaction medium
[21,24,25] In view of the strategic importance of the
oxyferro intermediate in the process of dioxygen activation,
the influence of the physiological redox partner, cytochrome
b5, on its autoxidative breakdown has been examined in
detail: though increasing the rate of regeneration of ferric
enzyme from oxygenated CYP2B4 by a factor of about 8,
reduced donor protein added to the assay mixtures failed to
undergo substantial reoxidation, suggesting the electron
carrier to act as an allosteric effector in this reaction [38,42]
In accord with this, both apocytochrome b5and
aporubre-doxin reportedly stimulate autoxidative transformation of
oxy CYP101 to the ferric state [43] Superoxide departing
from regenerated P450 has been found to serve as a source
of hydrogen peroxide usually generated during NADPH/O2
to generate the hydroperoxo-iron derivative (compound O;Scheme 2) Intermediacy of the end-on Fe(III)-OOH specieshas 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 byreacting ferrous CYP101 with KO2[57] or bioreduction ofoxyferrous CYP101 with putidaredoxin [58]
Unless the protonated peroxide complex decays in anonproductive mode to liberate ferric enzyme and H2O2[18], conversion to the actual oxidant proceeds with asignificant energy release of 50 kcalÆmol)1 [59] Whileacylation of the distal oxygen to make it a better leavinggroup prior to Fenton-type homolytic O-O bond rupturehas been vitiated owing to discrepancies between theory andmeasured data [60], the most favoured activation pathway isheterolytic 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 push effect of thethiolate ligand in P450s has been shown to promoteheterolytic cleavage of heme-bonded dioxygen by increasingelectron density at the iron atom [62–66] The electron-donating properties of the active-site thiolate of CYP101have been demonstrated to be enhanced by putidaredoxin-induced alterations in enzyme conformation [50–67].Attempts were made to characterize the P450 reactiveoxygen intermediate Thus, iodosylbenzene, a single-oxygendonor [68], as well as peroxides and peracids, acting asversatile O2surrogates in oxidative reactions [69–71], havebeen revealed to elecit spectral perturbations with P450sclosely resembling those of the green, high-valent FeOPor• +
species (compound I) of peroxidases, including the ligated, P450-like chloroperoxidase enzyme [72–75] Thesefindings lent credit to the notion, that an analogous keyoxidant might be operative in P450-catalyzed monooxy-genations, too, albeit there is a significant difference betweenP450 and peroxidase models regarding the displacement ofthe iron atom from the porphyrin plane, resulting in longerFe-O bond in the P450 active intermediate [76] Densityfunctional studies demonstrate that both enzyme systems,though looking very similar, behave like chemical chame-leons, in which small alterations in the environment cancause drastic changes in the reactivity of the active species[76] Further support in favour of the idea of the involve-ment of a high-valent iron-oxene in P450 catalysis camefrom experiments with metalloporphyrin models [5,6,77] Ofparticular importance, a green oxo-ferrylporphyrin p-cationradical intermediate could be isolated and spectrophoto-metrically and chemically characterized, that was capable of
thiolate-Fig 1 Effect of hydrogen ion concentration on the Hill interaction
coefficient n for oxygen binding Rabbit liver microsomal N-oxide
for-mation from N,N-dimethylaniline was measured in the absence (d) and
presence (s) of 490 l M CO Reproduced from [30] with permission.
Trang 4oxygen transfer reactions [78] Nevertheless, identification
of the [FeO]3+adduct by UV-visible spectroscopic analysis
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 water [81] in a 4-electron reductive
process [82], uncoupling being controlled by substrate
accessibility [83] In fact, the presence of substrate has been
shown to stabilize the active oxy complex produced with
CYP2B4 and organic hydroperoxide, and the protective
effect is intensified by cytochrome b5binding [84,85] Active
oxidant thus preserved is thought to promote hydrogen
transfer from substrate to initiate monooxygenation
(Scheme 1); this step, which proceeds with a remarkable
low free–energy barrier, has been suggested to be governed
by peripheral heme substituents in the P450 molecule [86]
Firm evidence for the nonconcerted 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; kH/kD 11) for C-H activation using
norbornane [87], diphenylpropane [91] or difluorocamphor
[92] as the hydroxylatable substrates and (e) the results from
investigations with radical clock 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 recombination step
[16,93]
Despite the apparent predominance of the hydrogen
transfer mechanism as the initial step in substrate
hydroxy-lation, electron transfer to generate a carbocation, followed
by capture of a hydroxyl anion has been discussed as an
alternative oxygenating principle [94,95] The net outcome
would be oxidation of an otherwise unactivated C-C single
bond Although cations may be the logic precursor for
certain substrates with low oxidation potentials, such a
pathway cannot be reconciled with the large KIE and
stereochemical scrambling detailed above To quantitatively
assess the significance of electron transfer in the transition
states of hydroxylation reactions, 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 from the large magnitudes of the
combined primary and secondary KIEs for hydrogen
abstraction, rotation in the enzyme pocket was faster than
its relatively slow reaction (< 106Æs)1) with the putativeiron-oxene species [97], while the lifetimes of carbon-centredradicals derived from a diverse set of substrates are on theorder of about 10)10s [98] Moreover, the randomness ofthe apparent intramolecular KIEs for unrearranged andrearranged alcohol products generated from enantiomericdideuteriomethyl substrate forms implicated that more thanone reaction channel existed [99] This concept wasreinforced when the KIEs for NADPH-and cumenehydroperoxide-driven N-demethylation of amitryptiline byCYP2D6 were found to be severely discrepant [100].Examination of the competitive intermolecular KIE forsulfoxidation/N-dealkylation reactions mediated by bacter-ial CYP102 hinted at the involvement of two distinctelectrophilic oxidizing species [101], as was also concludedfrom the intermolecular noncompetitive KIEs for a- andb-hydroxylation of fatty acids by CYP152 peroxygenaseisozymes [102]
Probing of the metabolism of norcarane by CYP2B4revealed the formation of a cation-derived rearrangementproduct not compatible with the hydrogen abstractionmechanism [103] The latter was also challenged by thefinding that evaluation of the metabolic transformation of aseries of cyclopropane derivatives by CYP2B4 gave unrea-sonably high rate constants for oxygen rebound (kOH)ranging from 1.5 to 7· 1012s)1; this disparate result wasrationalized by possible steric effects in the enzyme’s activesite causing overestimation of the kOHvalues [17] However,experiments on the CYP2B1-catalyzed hydroxylation of anew constrained substrate, that would be less likely to besubject to steric constraint, also yielded an incredibly highapparent kOHvalue of 1.4· 1013s)1[104] Moreover, theplot of the ratio of rearranged to unrearranged alcoholproducts vs the rate constant for rearrangement of theputative radical intermediate (kr) revealed a lack of corre-lation between these parameters [104] In addition, hyper-sensitive radical probe studies with four P450 isozymes gaveconsistently small amounts of rearranged products, ham-pered radical ring opening on steric grounds being unlikely[105] The sum of these findings thus suggested that therewas either an error in the kinetic scale for fast radicalreactions or the mechanistic paradigm of P450-mediatedhydroxylations was incomplete To solve this problem,further hypersensitive radical probe substrates were intro-duced, that could distinguish between radical and carbo-cation intermediates on the basis of the identity of therearranged products [9,106,107] Oxidation of these probeswith several members of the CYP2 family gave cation-derived rearrangement products, disproving the assumptionthat such rearrangements arose exclusively from radicalspecies Variable partitioning between the radical andcarbocation mechanisms thus was concluded to explainthe wide range of kOHvalues described above [106] Fromthe small amounts of radical rearrangement productsgenerated from the hypersensitive probes, the radicallifetimes in the P450-catalyzed reactions could be calculated
to range from 70 to 200 fs [106,107], which are too short fortrue radical intermediates, but rather correspond to vibra-tional lifetimes or the lifetimes of transition states Hence,the cationic intermediates observed could be ruled out tooriginate from oxidation of such transient radicals, so thattheir occurrence necessitated another mechanistic enigma
Trang 5In this regard, the most plausible premise is insertion of
OH+ into a C-H bond to generate protonated alcohol
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 3A) to release OH+and [FeO]+[106,107]
However, density functional analysis of mechanisms
involved in ethylene epoxidation by a Fe(III)–OOH model
disclosed barriers for the various pathways of 37–53 kcalÆ
mol)1[108] This was taken to indicate that
hydroperoxo-iron, as such, could not be the ultimate oxidant, in line with
its significant basicity and poor electron-accepting
capabil-ities [108] Moreover, molecular orbital calculations carried
out with a similar model system unveiled nonrepulsive
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/O2- and iodosylbenzene-dependent
meta-bolism of lauric acid by CYP2B4 favoured the Fe(III)-H2O2
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 idea:
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 diprotonated 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 in P450 catalysis is also corroborated by studies on
heteroatom oxidation Thus, comparative investigations
on the NADPH/O2- and cumene hydroperoxide-driven
N-hydroxylation of 4-chloroaniline by CYP2B4 indicated
discrepancies in the positions of the Soret maxima in the
absolute spectra of the individual oxy complexes [116]
Noteworthy, transformation of P450 to the denatured P420
form through treatment with either
p-chloromercuribenzo-ate or deoxycholp-chloromercuribenzo-ate rendered the hemoprotein a more
powerful peroxygenase [116], but disrupted NADPH-linked
monooxygenase activity [117] Hence, resonanace
stabiliza-tion via the thiolate push effect (see above) did not appear
to be obligatory when peroxide substituted for reducedcofactor and dioxygen While N-(4-chlorophenyl) hydroxy-lamine was found to be the major metabolic product undermixed-function conditions, a marked change to the prepon-derant formation of 1-chloro-4-nitrobenzene was observedwhen organic hydroperoxide served as the oxygen donor[116] Involvement in the N-oxidative process of CmO•
(CmOÆ2) radicals could be safely ruled out owing toinsensitivity of the reaction toward radical scavengers,whereas blockage of turnover by cyanide hinted at aniron-based mechanism [116] The sum of these findingsraised serious questions as to the commonness of theoxygenating species operative in the NADPH- and hydro-peroxide-sustained hydroxylations In fact, evidence hasbeen provided for the existence of fairly stable Fe(III)-OORintermediates generated by reacting organic hydroperoxideswith mononuclear iron catalysts [118–120] or intactCYP2C11 [121], and their ability to transfer oxygen tosubstrates prior to heterolytic cleavage at low temperatureshas been ascertained [122–124] As N-hydroxylation of4-chloroaniline by the putative Fe(III)-OOR species mustcompete not only with conversion of the intermediate to[FeO]3+, but also with self-destructive oxidation of the hememoiety of P450 [125], it seems worth mentioning that therate of cumene hydroperoxide-induced loss of CO-reactiveCYP2B4 [85] could be demonstrated to be far below that ofrelease of N-oxy product from the ternary complex [116].There is also reason to envisage iron-bound hydro-peroxide as a potential oxidant in NADPH-promotedN-oxygenation of N,N-dimethylaniline by CYP2B4: thepresence of superoxide dismutase inhibits the reaction by75%, whereas catalase or mannitol leave N-oxide formationunaffected, dismissing free H2O2or OH•
radicals to act ascatalysts [126] Notably, investigations with a superoxide-generating system ruled out O2• )itself to function as theactive intermediate, so that superoxide was invoked to serve
as a source for production of the ultimate oxygenatingspecies, presumably Fe(III)-OOH, catalyzing attack on theelectron-rich nitrogen centre of the tertiary arylamine[126–128] The active oxidant thus was anticipated to arisefrom interaction, in the presence of protons, of newlygenerated O2• )with either ferrous or oxyferrous [Fe(III)-
O2• )] P450, as given in Eqns 1 and 2 [129–132] That OOH generated in this way would only serve as a precursor
Fe(III)-in the transformation to:
FeðIIÞ þ OÆ2 þ Hþ! ½FeðIIIÞ OOH ð1Þ
½FeðIIIÞ OÆ2 þ OÆ2 þ Hþ! ½FeðIIIÞ OOH þ O2
ð2Þiron-oxene as the actual catalyst could be discounted
on kinetic grounds As an example, the reactionsequence given in Eqn 2 follows second-order kineticswith a rate constant of 4· 103
M )1Æs)1 [133], whileinjection into Fe(II)-O2 of the second electron toproduce compound I during regular catalytic cycling is
a diffusion-controlled process characterized by a rateconstant of 4· 1010
M )1Æs)1[134] Comparison of thesedata no doubt precludes the major portion of ferrylmaterial required for efficient substrate turnover tooriginate from the dismutation-type bypass reaction As
SCys
O H
Fe3+ O
SCys
OH H
Fe3+ O
SCys
OH H H
Scheme 3 Potential second oxidant species in P450 catalysis Data
adapted from [108] with permission.
Trang 6conversion 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 hydroxylations came from studies with
mutated P450s The crystal structures of bacterial CYPs 101,
102 and 108 contain a highly conserved active-site threonine
within H-bonding distance to the peroxo-iron unit [135] Of
particular interest, attenuated camphor and laurate
hydroxy-lation was observed, when T252/268 in the CYP101 and
CYP102 polypeptide, respectively, were replaced with
alan-ine [136,137] Nevertheless, the T252A variant was found to
accept electrons from NADH and reduce dioxygen to H2O2
[137] via the intermediacy of hydroperoxo-iron [53]
Muta-tion was considered to disrupt a key step in H+delivery,
presumably introduction of the second proton to hamper
O-O bond dissociation [53] Therefore, P450 mutants devoid
of the active-site threonine were regarded ideal means for
testing the direct involvement of hydroperoxo-iron in
epoxidations Indeed, a drastic increase 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 alkene metabolism at an increased ratio of
epoxidation to 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 of probes designed to
give different rearrangement products with radical and
cationic intermediates [99,105,107,140] Moreover,
trun-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 mutant of CYP102: the engineered enzyme
fostered sulfoxidation of p-(N,N-dimethylamino)thioanisole
relative to N-dealkylation of the 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 hydroperoxo-iron-promoted mechanism [142]
However, when the measurements were conducted withiodosylbenzene in place of NADPH/O2to directly generatethe favoured [FeO]3+entity [68], the enzyme variant stillmediated N-oxygenation of the tertiary arylamine at a rateless than half that of the wild-type-catalyzed reaction [142],
so that reasonable interpretation of the data seems difficult
Evidence from comparative studies with non-P450hemoproteins and metalloporphyrin modelsHemoglobin (Hb) and myoglobin (Mb) When operative
in its natural environment, the erythrocyte Hb exerts like monooxygenase activity [143] in that the concurrence ofmultiple reductase systems permits NAD(P)H-supportedelectron transfer to the pigment [144,145] In fact, iso-lated Hb reconstituted with NADPH-cytochrome P450oxidoreductase (EC 1.6.2.4) has been demonstrated to bringabout NADPH/O2-promoted alkane hydroxylation [146] aswell as N- and O-dealkylation reactions [147], albeit atconsiderably lower catalytic potency as compared with P450enzymes Of particular interest, Hb has been found tomediate formation of p-aminophenol from aniline both inintact erythrocytes, supplemented with glucose to allowNADPH production via the pentose phosphate pathway[148], and in a reconstituted system containing P450reductase, NADPH and atmospheric oxygen [149] In thelatter case, catalase inhibited enzyme activity by about 94%
P450-in the absence or presence of reductase, suggestive of
a hypothetical mechanism for p-hydroxylation of thearomatic amine involving H2O2, formed throughdismutation of autoxidatively generated superoxide, toproduce the active intermediate, Hb(III)-OOH [149] Inline with this, alkaline hemin (ferriprotoporphyrin IX) hasbeen shown to activate O2to the hydroperoxide anion in thepresence of NAD(P)H [150] with the immediate insertion
of oxygen into the benzene ring of aniline to yieldp-aminophenol [151] Formation of the oxygenatingspecies has been recognized to be facilitated by the binding
to HbO2 of aniline and some of its derivatives, causingdistortion of the iron-oxygen bond to such an extent as toaccelerate autoxidation by alleviating electron transfer fromferrous iron to O2 [147,152] Superoxide displaced fromHbO2has been postulated to contribute to production ofthe hydroperoxo-methemoglobin entity by reducing heme-bound oxygen in the presence of a proton source as given inEqn 2 [131,153] Indeed, radiolytically reduced samples ofoxygenated Hb [154,155] and Mb [156–158] at cryogenictemperatures have been shown by EPR studies to generatethe peroxo-bound hemoproteins, with the Fe-O-O unitbeing stabilized by bonding to the distal histidine proton; thelatter was detected to be transferred upon annealing to givethe hydroperoxo derivatives Similar results were obtained,when metmyoglobin was reacted with H2O2at 77 K [159].The ability of Hb to catalyze heteroatom oxygenation iswell established Thus, methemoglobin and H2O2transformthianthrene 5-oxide to both the 5,5-and 5,10-dioxidemetabolites [160] The finding that most, if not all, of thesulfoxide oxygen in the 5,5-dioxide product originates fromhydrogen peroxide but not from18O2has been rationalized
by the possible participation in this reaction of a iron catalyst This view is compatible with the capability ofmononuclear peroxo intermediates derived from iron and
Trang 7peroxo-titanium porphyrin complexes upon treatment with
super-oxide and H2O2, respectively, to directly promote
sulfoxi-dation reactions [161]
Similarly, Hb has been reported to perform
N-hydroxy-lation of 4-chloroaniline both in erythrocyte suspensions
[162] and in aerobic systems reconstituted with either
NADPH-P450 reductase or the NADH-cytochrome b5
reductase/cytochrome b5 segment of the electron transfer
chain in the presence of NAD(P)H [163,164] Under these
conditions, addition to the assays of superoxide dismutase or
catalase disrupted N-oxygenating activity by about 70%,
again posing emphasis on the pivotal role of H2O2in forming
the active oxidant It should be noted that N-oxidative
metabolism of 4-chloroaniline is associated with optical
changes characterized by a Soret band at 418 nm in the
absolute spectrum [163], closely resembling the spectral
perturbations arising from reduction of MbO2by hydrated
electrons [134] or reaction of ferrous CYP101 with
super-oxide [57] to yield 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 also apply to one-electron oxidation of the
nitrogen centres in N,N-disubstituted p-phenylenediamines
to give Wurster’s blue aminyl radicals: the HbO2-dependent
processes have been shown to be decelerated by up to 50%
in the presence of catalase, whereas superoxide anion was
likely to be of minor importance in 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 evidence for a vital role of autoxidatively
liberated H2O2 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 represent a
transient intermediate in the production of oxygenating
ferryl material However, it seems improbable that the route
of ferryl-Hb formation should be preferentially via the
peroxide shunt: the sluggish autodecomposition of HbO2
(k 10)3M )1Æs)1) in the presence of the anilines [166] to
finally yield H2O2together with the relatively low rate of
peroxide association with ferric globin (k¼ 4.8 ·
102
M )1Æs)1) [159] undoubtedly impose considerable
con-straints on the overall rate of compound I formation,
whereas its direct generation upon electron introduction
into the oxyferrous entity is a very rapid process as oulined
above [134] The sum of these findings refutes compound I
to contribute to significant extent to the total amount of
hydrogen peroxide-induced active oxidant, but rather
favours Hb(III)–OOH itself or the iron(III)-H2O2 adduct
[110] to serve in this function (Scheme 3) This view is
endorsed by the fact that imidazole, acting as the proximal
axial ligand in electron-rich iron-porphyrin model
com-pounds, appears to prolong the lifetime of the H2O2-derived
hydroperoxo-iron species, such as to permit direct oxygen
insertion into substrate [167] In accord with 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; EC1.14.99.3) is distinct from P450s, the reactions catalyzed bythis enzyme are, nevertheless, part of the same heme-dependent reaction manifold that underlies the catalyticaction of all hemoproteins The first metabolic processmediated hy HO is self-hydroxylation of heme to forma-meso-hydroxyheme (Scheme 4), using the histidyl-ligatedheme group as both a prosthetic unit and substrate[125,169–171] Whereas transformation by plant [172] andbacterial [173] HO enzymes requires electron supply by anNADPH-ferredoxin reductase/ferredoxin couple analogous
to mitochondrial and microbial class I P450s, mammalianheme oxygenases accept reducing equivalents, in thepresence of dioxygen, from NADPH-P450 reductase,resembling microsomal class II P450s with respect to theirability to functionalize unactivated C-H bonds [170] There
is strong evidence for Fe(III)-OOH to act as the hydroxylating species in HO catalysis Thus, H2O2has beenfound to be able to replace NADPH/O2 in supportingthe first step in heme oxidation, while ferryl-formingacyl hydroperoxides were incompetent [174] Moreover,application of ethyl hydroperoxide as the oxidant could bedemonstrated to promote generation of a-meso-ethoxyheme[175] Studies with the four meso-methylmesohemeregioisomers disclosed the electron-donating methylsubstitutents to govern the regiochemistry of meso-hydroxylation on an electronic rather than steric basis,implicating electrophilic addition of the oxygen to theporphyrin ring [176,177] It should be mentioned, in thiscontext, that the H39V mutant of rat outer mitochondrialmembrane cytochrome b5has been shown to be capable ofbuilding a coordinate ferric hydroperoxo intermediate uponreduction of the oxyferrous complex with hydrazine, whichadds a hydroxyl group to the porphyrin to produce meso-hydroxyheme [178]
meso-Direct evidence for the occurrence of Fe(III)-OOHduring normal catalytic turnover of HO was furnished byEPR and ENDOR 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 tion between the a-meso-carbon of the porphyrin moiety
forma-Scheme 4 Proposed mechanism of HO-catalyzed conversion of hemin
toa-meso-hydroxy-heme The heme unit is shown in a truncated form.
Reproduced from [125] with permission.
Trang 8and the terminal oxygen atom of the hydroperoxo entity;
this process was activated by delivery of the second proton
by a carboxyl donor, presumably D140 [180,181] Finally,
optical absorption and EPR measurements permitted the
detection of a hydroperoxo intermediate derived from a
synthetic Fe(III)-porphyrin complex, with electrophilic
addition of the axially ligating OOH– 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 subsequent steps of peptic
and tryptic digestion of 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 attractive model
for studying P450-type oxygen transfer reactions [184]
Thus, addition of ascorbate to MP8/H2O2-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 increased; this was attributed to
involve-ment in catalysis of a (hydro)peroxo-iron intermediate
[185,186] In accord with this, NADPH/O2-sustained
conversion of aniline to p-aminophenol by heme-peptide
reconstituted with NADPH-P450 reductase has been
shown to be highly susceptible to the presence of
catalase [187] Moreover, 18O-labeling experiments with
MP8 in the presence of ascorbate revealed the biocatalyst
to p-hydroxylate aniline with full transfer of oxygen from
H182O2, while rapid exchange of the labelled oxygens of
H2O2 with unlabelled H2O occurred, pointing at
reversi-bility of formation of the high-valent iron-oxene species to
produce a porFe(III)-H2O2complex [188] Similarly, water
has been advocated to play a decisive role in regeneration,
through reaction with (R• +)MP8Fe(IV)¼ O, of the active
oxidant operative in the microperoxidase/H2O2-drivenhydrocarbon oxygenation in bi- and tricyclic aromaticcompounds [189] or oxidative aromate dehalogenation[185,190] (Scheme 5) It should be emphasized thatreaction of iodosylbenzene with purified CYP2B1 [191]
or nonporphyrin iron(III) chelates in basic media [132,192]has been detected to prompt O-O bond formation at the ironcentres Alkoxylating dehalogenation of halophenols,carried out by MP8/H2O2in alcoholic solvents, has beenhypothesized to implicate a mechanism, in which the iron-oxene resonance form reacts with alcohol to generate anFe(III)-OOR intermediate [193] Unequivocal indenti-fication of the MP8-based (hydro)peroxo-iron(III) entityhas been achieved by optical absorption [194] and rapid-freeze EPR measurements [195]
Synthetic metalloporphyrin models Synthetic porphyrins (Fig 3) were selectively tailored as models ofthe P450 active site to gain more detailed insight into themechanistic basis of oxygen transfer reactions Using a set
metallo-of meso-tetraarylporphyrine derivatives (Fig 3A), stilbene was found to be subject to H2O2-sustainedconversion to the oxide metabolite in aprotic solvent withtrace amounts of allylic oxidation products, ruling out iron-oxene or OH radicals to be responsible for olefinepoxidation, while hydroperoxo-iron was likely to bethe active oxidant [124,167] Similarly, the lack of
cis-18O-incorporation, at low temperature, from labeled waterduring perbenzoic acid-supported epoxidation ofcyclooctene by the polyhalogenated TPFPP analogue wasinterpreted to mean that the electronegatively substitutediron porphyrin generated a relatively stable Fe(III)-OORspecies, which directly transferred its oxygen to the olefin[123] This postulate is in line with the observation that there
is a strict relationship between the selectivity ofnorbornylene over a-methylstyrene epoxidation by theTDCPP-porphyrinato-iron complex and the structure ofthe peracids used [196] The same principle also applies tothe cyclooctane vs cyclooctene oxidation catalyzed by athiolate-ligated meso-tetraarylporphyrin model (Fig 3B)with various p-substituted perbenzoic acids [197]
Fig 2 Chemical structure of microperoxidase-8 Data taken from
H2O H+
F–
N
H 3 C
O
Scheme 5 Proposed reaction mechanism for a MP-8-catalyzed genation pathway.
Trang 9dehalo-Moreover, acylperoxo-iron(III) has been claimed to also
serve as the effective catalyst in the peracid-driven
cyclohexane hydroxylation depending on the nature of the
anionic axial ligands of the Fe(TPFPP) adduct [198]
Studies with a second genre of metalloporphyrin systems,
containing molecular oxygen and a coreductant such as
ascorbate to mimic 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 complex in the
ascorbate/O2-driven process [199]
The reactive (hydro)peroxo-metalloporphyrin species in
the above model systems have been characterized by visible
spectroscopy, NMR, EPR, and Mo¨ssbauer data upon
combination of 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 geometry of the transient
reduced ferrous dioxygen form, calculated by applying
nonlocal DFT methods, indicated an asymmetric end-on
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 regio-and stereospecific cis-dihydroxylation of aromatic
rings, initiating aerobic degradation of aromatic compounds
in soil bacteria, and are targets for bioengineering in
bioremediation [204] They are a consortium of two or
three protein components involving a Rieske Fe2S2cluster to
channel electrons from NAD(P)H via a flavin-containing
reductase to a mononuclear iron centre; the latter is believed
to be the site of dioxygen and substrate activation [204] This
electron transfer chain thus functions like the heme centres in
class I P450s acting in unison with their associated sulfur redox partners [135] Analogous to the role ofputidaredoxin as an effector in CYP101 catalysis [50,67],binding of phthalate oxygenase reductase (EC 1.18.1), aflavo-iron-sulfur polypeptide, to phthalate dioxygenase(PDO; EC 1.14.12.7) has been advocated to tune theenzyme’s structure for oxygenating activity on an allostericbasis [205] Moreover, toluene dioxygenase (TDO;
iron-EC 1.14.12.11) and naphthalene 1,2-dioxygenase (NDO;
EC 1.14.12.12) have been found to mediate P450-likemonooxygenations when provided with appropriatesubstrates [206,207]
Availability of the crystal structure of NDO as well asspectroscopic data provide a rationale for the catalyticmechanism of this class of enzymes Naphthalene 1,2-dioxygenase is a heterohexamer composed of an equimolarcombination of a- and b-subunits, each a-subunit bearing
an Fe2S2 cluster and a mononuclear iron site [208] Twohistidines and one bidentate aspartate ligand, the socalled
2-His-1-carboxylate facial triade, encountered with variousnonheme iron, oxygen-activating enzymes, occupy one side
of the mononuclear iron coordination sphere [209] strate binding to produce an open coordination position onFe(II) has been suggested to be critical in O2-activation,allowing two-elctron transfer from both the mononucleariron centre and the reduced Rieske cluster to generate anFe(III)-OO(H) intermediate [210,211]; the latter has beenproposed to exert a concerted mode of attack on substrate,explaining the cis-specificity of the dihydroxylation reaction
Sub-In accord with this concept, hydrogen peroxide has beenreported to be able of substituting for NAD(P)H/O2 inNDO-dependent cis-dihydrodiol formation, both oxygenatoms in the product deriving primarily from H2O2[212].Moreover, benzene has been demonstrated to act as both asubstrate and an uncoupler of NDO, causing the release of
H2O2during the reaction [213] More recently, the role of aputative end-on (hydro)peroxo-iron catalyst in NDOturnover has been ascertained by the detection, in theenzyme’s crystal structure, of an indole-oxygen adductbound to the mononuclear iron [214] Circumstantial EPR
N N
Cl F
F F F
F F
F
N
N N
N
Fe+++
HN
O O S
N N
Cl –
TDCPP
TPFPP TDFPP
S
H H H
Fig 3 Chemical structures of
iron(III)por-phyrin complexes used as P450 model species.
Data taken from [167,197,202, 203].
Trang 10spectrometric 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 terminal oxygenase, lent
further support to the idea of peroxo-iron-sustained
oxygenation chemistry [215,216] Apart from
O-demethy-lation, PMO can functionalize aliphatic and aromatic C-H
bonds, with H2O2 being liberated in the presence of
uncoupling compounds [217,218] By a substrate-modulated
reaction, PMO has been demonstrated to also act as a
peroxotransferase: using vinylbenzoate 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 18O2 [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 proceed along a common track, with
Fe(III)-OO(H) serving as the 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 enhanced by results obtained
with iron-based functional 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 and steric properties of the ligand
environ-ments has been recognized to afford high-spin
hydroper-oxo-iron species in combination with H2O2, 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 equivocal in view of the nearly insignificant amount
of 18O-incorporation from water into the diol products
[225]
Bleomycin and related metal-based model complexes Thebleomycins (BLMs) constitute a family of naturalglycopeptide antibiotics produced by the fungusStreptomyces verticillus, which are used as antineoplasticagents owing to their ability to degrade DNA uponbioactivation in the presence of appropriate metal ionsand a source of dioxygen [226] Although iron appears to bethe most effective BLM cofactor, other metals also bindstrongly to the antibiotic [227] A key to the uniquereactivity of the nonheme iron(II) site of BLM (Fig 5)toward O2seems to reside in one of its equatorial ligands,the pyrimidine moiety [228] The mechanism of oxygenactivation is strongly reminiscent of P450 cycling [226],the ferric intermediate being reduced either chemically bydithiothreitol and ascorbate [229] or enzymatically byNADPH-P450 reductase [230] Oxygen surrogates such asiodosylbenzene [229], H2O2 or alkylhydroperoxides [231]have been shown to be apt to bypass reductive
O O
Fe 3 +
O O–
Fe 3 +
O O
Fig 4 Iron-bound ligand frameworks used as models of dioxygenases Data taken from [224].
Trang 11O2-activation The architecture of activated BLM has been
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 of the DNA deoxyribose sugar, for which multiple
scenarios have been set forth including hetero- or homolytic
O-O bond cleavage of activated BLM to yield a high-valent
oxo-iron species affording DNA attack, or direct reaction of
HOO-Fe(III)BLM with its target to give a DNA radical,
H2O, and an Fe(IV)¼ O entity [226,227,239,240] Among
these possibilities, the homolytic pathway, entailing the
production of undiscriminating free OH radicals, could 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 to be energetically unfavourable by at least
40 kcalÆmol)1, which is more than 150 kcalÆmol)1less likely
than for P450 [223,241]; this was attributed to differences in
the nature of 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 to considerably increase electrophilicity of
the oxidant [223,241] Experiments with HOO-Co(III)BLM
green, a stable analogue of activated BLM, provided a rare
snapshot of a reactive intermediate poised to initiate the
hydrogen atom abstraction event: the distal oxygen of the
hydroperoxide is only 2.5 A˚ away from the C4¢-H of
cytosine [242] Further information was gained from studies
with synthetic iron complexes assumed to be model systems
for BLM because of their capabilities to inflict DNA strand
scission in the presence of ascorbate/O2 [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 H2O2was the oxidant in place
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 dation of alkenes [229,236,245] Noteworthy, experiments onBLM-mediated epoxidation of cis-stilbene, employing iod-osylbenzene as the oxygen donor in the presence of H182O,disclosed the epoxide oxygen to derive primarily from labeledwater [229,245] As pre-equilibration of the oxidant withwater prior to activation has been demonstrated not to result
epoxi-in exchange with H2O [229,246,247], this observationstrongly suggests O¼ Fe(IV)BLM• + to be capable ofundergoing exchange with solvent to regenerate Fe(III)-OOH as the epoxidizing species [132] This view is substan-tiated by studies with the iron complex of the BLM mimicdepicted in Fig 6A Here, iodosylbenzene in basic mediagives rise to formation of a hydroperoxo intermediatepresumed to be the catalyst in the oxidative turnover ofolefins [192] Hydrogen abstraction from methanol, used asthe solvent, to produce a radical intermediate has beenascertained to result from direct reaction with the alcohol ofthe hydroperoxo unit formed by combination of H2O2withthe iron-ligated BLM model shown in Fig 6B [248]; themethoxy radical thus generated has been found to induceligand modification through attack on the carbon a to theamidate
A plethora of bioinspired ferric (hydro)peroxo complexeswith the peroxide ligand being bound in a side-on (g2) orend-on (g1) fashion have been created as more generalmodels of metalloenzymes by reacting H2O2or ROOH withvarious iron chelates [223,249] While the side-on peroxo
Fig 5 Chemical structure of iron bleomycins.
Data taken from [240].
NH NH
HN HN
N N
N N
N
Br H
N
N N
N
N
N
N N
N
N
N N
D
Fig 6 Nitrogenous ligands used to construct the corresponding iron complexes as mimics of bleomycin and mononuclear nonheme metallo- enzymes Data taken from [192,248,252,254].
Trang 12entity is relatively inert toward 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 Fig 6C–F Similarly,
Cr-OOH, produced with a macrocyclic chromium-based
system (Fig 6C), exhibits H+-assisted oxidative reactivity
toward triarylphosphines [256] Generally, the
peroxide-driven route appears to be under stereochemical control
exerted by a-substituents [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, in the presence of atmospheric
dioxygen, of a relatively labile Fe(II) complex such as bis
(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 to 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/O2 adduct [258]
analogous to P450 chemistry, so that more concerted redox
steps can be envisioned Substrate transformation posits the
participation of structurally similar hydroperoxo-iron
cat-alysts with different formal oxidation states [258,260], the
process being driven to exothermicity via water formation
(Eqn 3)
RHþ PhNHNHPh þ O2! ROH þ PhN ¼ NPh þ H2O
ð3ÞElectrochemical investigations support this concept,
revealing autoxidation of diphenylhydrazine, when
exposed to O2, to liberate hydrogen peroxide, which
collapses with iron(II) to give an Fe(II)-H2O2 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 the pregnene nucleus, followed by oxidative
acyl-carbon cleavage 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 of18O2permitted analysis of
the pattern of isotope incorporation into the reaction
products, best rationalized by invoking the participation of
a nucleophilic peroxo-iron species in the C-C bond fission
process [263,264] It has been hypothesized that hydroxy-progesterone binds to unprotonated CYP17 such
17a-as to interrupt a proton shuttle to Fe(III)-OO–, facilitatingC-17 side-chain dissociation through peroxide chemistry[265] Employing recombinant CYP17 and labeled hydroxy-androstene-17b-carbaldehyde, a pregnenolone analogue, asthe substrate in combination with18O2, isotope-partitioningexperiments suggested androgen genesis to be closely linked
to formation of an iron-peroxy adduct prone to tation [266] A similar paradigm also appears to apply to thefinal step in aromatase (CYP19)-catalyzed biotransforma-tion of androgens to estrogens [267] As illustrated inScheme 7, this event entails aromatization of the A-ring
fragmen-of androstenedione via oxidative decarbonylation fragmen-of the19-aldehyde intermediate to release formic acid with theconcomitant production of estrone [268] When humanplacental microsomes fortified with deuteriated androgenprecursors in the presence of18O2were used to explore themechanistic course of aromatization, a transient ferriperoxy-hemiacetal-like complex (Scheme 7) turned out to
be a strong contender to explain the step of C-C bondscission [269,270] This type of nucleophilic attack on acarbonyl group by peroxo-iron has also been evidenced inthe final C-C bond cleaving process during sterol biosyn-thesis in the lanosterol 14a-demethylase (CYP51) reactioncascade [271,272]
Of particular interest, the NADPH/O2-dependent version of cyclohexane carboxyaldehyde to cyclohexene byreconstituted CYP2B4 has been evaluated as a potentialmodel for deformylation reactions brought about bysteroidogenic P450s: mass-spectral analysis unveiled for-mate to be formed in about an equimolar amount withrespect to olefinic product [11] Similarly, a series of otherxenobiotic C-5 aldehydes have been shown to be deform-ylated to variable extent by highly purified rabbit liver P450s[273] Moreover, externally added H2O2in place of the usual
con-O2-reducing system has been reported to be active withCYP2B4 in supporting deformylation of aldehydes [11,274].Employing 3-phenylpropionaldehyde as the substrate, anadduct was detected with a mass corresponding to that ofnative heme modified by a phenylethyl group, presumablyarising from the reaction of a peroxo-iron entity with thealdehyde to give a peroxy-hemiacetal [274]
Evidence from experiments with genetically engineeredP450 enzymes and molecular modelling
Taking advantage of the debilitating effect on protondelivery to Fe(III)-OO–exerted by mutagenesis of the highlyconserved active-site threonine in P450s (see above),experiments conducted with the T306A mutant of CYP17disclosed an about sevenfold increase in the proportion ofacyl-carbon cleavage vs hydroxylation activity duringandrogen biosynthesis as compared with the wild-typeenzyme [275] This was taken to substantiate the assumption
of juxtapositioning of the nucleophilic ferric peroxide anionand the carbonyl group of the substrate to be a compulsoryprerequisite for directing the enzymatic flux toward C-Cbond rupture Similarly, replacement of glutamate atposition 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
Trang 13model constructed by alignment of the CYP19 sequence
with the known crystal structures of bacterial P450s, E302
was postulated to be essential to activation of the 19-oxo
group of the substrate for 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 the 2b-hydrogen in the
A-ring of the C-19 substrate and donation of a proton to the
3-keto entity, respectively, to permit enolization [277,278]
(Scheme 7) A threonine switch, 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 were ambiguous [277] In fact, switching from
iron-oxene to peroxo-iron chemistry through threonine-302 to
alanine mutagenesis of truncated CYP2B4 could be
dem-onstrated by studies comparing the catalytic specificity of
deformylation of cyclohexane carboxaldehyde with that of
hydroxylation of other compounds [279] Moreover,
inves-tigations on the mechanism-based destruction of CYP2B4
by aldehydes revealed augmented 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 insignificant sequence identity with P450s in
the heme-binding region [282], but bearing a C-terminal
flavoprotein fragment in the single polypeptide chainstructurally resembling NADPH-P450 reductase; the latter
is separated from the heme domain by a calmodulinconsensus binding sequence [283] Importantly, NOSenzymes are dimeric proteins, in which flavin-to-hemetransfer of electrons provided by NADPH proceedsexclusively between adjacent subunits in the heterodimer,implying domain swapping for proper alignment of thereductase and oxygenase entities [284] Tetrahydrobiopterin(BH4), located close to the heme unit [285], has been shown
to contribute to stabilization of the NOS dimers [286].Moreover, the modifier binds cooperatively to the substrate-binding region [287] and facilitates electron flow tooxyferrous NOS [288]
The physiological role of NOS pertains to the tion of NO•
produc-, an important signaling moleculeproduc-, and citrullinethrough oxidative degradation of NG-hydroxy-L-arginine,generated via primary N-hydroxylation of one of the twoequivalent guanidino nitrogens of arginine [281] While
L-arginine and the homo-L-arginine derivative have beenoriginally thought to be the only true NOS substrates,more recent studies unveiled a series of N-aryl-N¢-hydroxy-guanidines to serve as NO•
donors after oxidative tion [289] Circumstantial analysis of the stoichiometry ofthe NO-forming reaction disclosed a three-electron process,with decomposition of the N-hydroxyarginine intermediateconsuming only 0.5 equivalents of NADPH per mol of O2
activa-during nitroxyl radical ejection [290] Comparative studieswith microsomal P450s [291,292] and biopterin-free as well
as BH4-containing NOS [293], exhibiting product ity with respect to the almost exclusive, superoxidedismutase-insensitive generation of equimolar amounts ofurea and NO•
selectiv-from arginine and some non-a-amino-acid
Scheme 7 Postulated final oxidation step in
aromatization catalyzed by CYP19 Data
adapted from [268] with permission.