DANICA GALONI´ C FUJIMORI,§
CHRISTOPHER T. WALSH,*,§AND
J. MARTIN BOLLINGER, JR.*,†,‡
Department of Biochemistry and Molecular Biology and
Department of Chemistry, The Pennsylvania State University,
University Park, Pennsylvania 16802, and Department of
Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, Boston, Massachusetts 02115
Received March 15, 2007
High-valent non-heme iron–oxo intermediates have been proposed
for decades as the key intermediates in numerous biological
oxidation reactions. In the past three years, the first direct
characterization of such intermediates has been provided by studies
of several RKG-dependent oxygenases that catalyze either hydroxyl-
ation or halogenation of their substrates. In each case, the
Fe(IV)–oxo intermediate is implicated in cleavage of the aliphatic
C–H bond to initiate hydroxylation or halogenation. The observa-
tion of non-heme Fe(IV)–oxo intermediates and Fe(II)-containing
product(s) complexes with almost identical spectroscopic param-
eters in the reactions of two distantly related RKG-dependent
hydroxylases suggests that members of this subfamily follow a
conserved mechanism for substrate hydroxylation. In contrast, for
the RKG-dependent non-heme iron halogenase, CytC3, two distinct
Fe(IV) complexes form and decay together, suggesting that they
are in rapid equilibrium. The existence of two distinct conformers
of the Fe site may be the key factor accounting for the divergence
of the halogenase reaction from the more usual hydroxylation
pathway after C–H bond cleavage. Distinct transformations cata-
lyzed by other mononuclear non-heme enzymes are likely also to
involve initial C–H bond cleavage by Fe(IV)–oxo complexes, fol-
lowed by diverging reactivities of the resulting Fe(III)–hydroxo/
substrate radical intermediates.
A large, functionally and mechanistically diverse family
of enzymes utilize similar, mononuclear non-heme Fe(II)
centers to couple the activation of oxygen to the oxidation
of their substrates.1–3In most cases, oxygen is inserted
into an unactivated C–H bond of the substrate (hydroxyl-
ation), but many other outcomes, including halogenation,
desaturation, cyclization, epoxidation, and decarboxyla-
tion, are known.3,4Each of these reactions is a two-
electron oxidation. The remaining two reducing equiva-
lents required for the four-electron reduction of oxygen
are often provided by a cosubstrate. The reducing cosub-
strates used by various family members include R-keto-
glutarate (RKG) (in the RKG-dependent enzymes3), tet-
rahydrobiopterin (in the pterin-dependent aromatic amino
acid hydroxylases5), reduced nicotinamides [in the Rieske
dioxygenases and (S)-2-hydroxypropylphosphonic acid
epoxidase1], and ascorbic acid (in 1-aminocyclopropane
1-carboxylic acid oxidase6). A few of the enzymes oxidize
their substrates by four electrons and thus do not require
a reducing cosubstrate. This subset includes the extradiol
dioxygenases,1isopenicillin N-synthase (IPNS),7and two
enzymes, 4-hydroxymandelate synthase and (4-hydroxy-
phenyl)pyruvate dioxygenase (HPPD), which effect distinct
four-electron oxidations of their common substrate.8The
latter two reactions are mechanistically similar to those
catalyzed by the RKG-dependent enzymes, because both
involve oxidative decarboxylation of an R-keto acid moiety
to provide two electrons.
This remarkable array of oxidative transformations is
made possible by the tuning of a largely conserved
mononuclear non-heme iron cofactor unit, which is
coordinated by as few as two protein ligands and thus has
as many as four sites available to coordinate substrates.
In the most common coordination sphere, three protein
ligands of a (His)2-(Asp/Glu) motif, known as the “facial
triad” because they occupy one face of an octahedron,
leave three remaining sites on the opposite face for
substrate coordination.9Reaction mechanisms proposed
for these enzymes have invoked several intermediates
following the addition of oxygen to the Fe(II) center.1,2
Two types of intermediates have been proposed: Fe-
coordinated (su)peroxo complexes with an intact O–O
bond, [Fe–O2]2+/3+, and high-valent Fe(IV)–oxo interme-
diates [or even Fe(V)–oxo for the Rieske dioxygenases],
[FedO]4+/5+. In particular, the high-valent Fe–oxo inter-
mediates have been suggested to initiate substrate oxida-
tion. In most cases, activation of the substrate involves
abstraction of the H-atom of the target C–H bond by the
Fe(IV)–oxo intermediate to yield a substrate radical and
Carsten Krebs obtained his Ph.D. in 1997 from the Ruhr-Universität Bochum and
in the Department of Biochemistry and Molecular Biology and the Department
Danica Galonic ´ Fujimori obtained her Ph.D. in 2005 from the University of Illinois
in 2005. She received a postdoctoral fellowship from the Damon Runyon Cancer
Christopher T. Walsh is the Hamilton Kuhn Professor of Biological Chemistry
and Molecular Pharmacology at Harvard Medical School. His research focuses
of Medicine, and the American Philosophical Society.
* To whom correspondence should be addressed. C.T.W.: Department
of Biological Chemistry and Molecular Pharmacology, 240 Longwood Ave.,
Boston, MA 02115; phone, (617) 432-1715; fax, (617) 432-0438; e-mail,
email@example.com. J.M.B.: Department of Biochemistry
and Molecular Biology, 208 Althouse Laboratory, University Park, PA
16802; phone, (814) 863-5707; fax, (814) 863-7024; e-mail, firstname.lastname@example.org.
C.K.: Department of Biochemistry and Molecular Biology, 306 S. Frear
Laboratory, University Park, PA 16802; phone, (814) 865-6089; fax, (814)
863-7024; e-mail, email@example.com.
†Department of Biochemistry and Molecular Biology, The Pennsyl-
vania State University.
‡Department of Chemistry, The Pennsylvania State University.
§Harvard Medical School.
and Molecular Biology and Chemistry at The Pennsylvania State University. His
research program focuses on the use of kinetic and spectroscopic methods to
Acc. Chem. Res. XXXX, xxx, 000–000
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an Fe(III)–OH complex (Scheme 1). The so-called oxygen
rebound, which was originally proposed for heme en-
zymes10and formally involves recombination of a coor-
dinated hydroxyl radical equivalent with the substrate
radical, yields the hydroxylated product and a coordina-
tively unsaturated Fe(II) center. In addition to substrate
hydroxylation, many other outcomes following H-atom
abstraction by the Fe(IV)–oxo intermediate are docu-
mented. These include transfer (formally as the radical)
of a ligand of the Fe center to the substrate radical.
Examples include transfer of a halogen atom in the RKG-
dependent halogenases4and transfer of a thiyl group in
Alternative reactivities that do not involve radical
recombination with a ligand include desaturation and
cyclization of the substrate. Formally, these reactions
involve abstraction of a second H-atom by the Fe(III)–OH
complex to yield the desaturated or cyclized product and
an Fe(II)–OH2complex. However, other pathways are
possible, making delineation of the mechanisms of these
alternative outcomes a high priority for future studies.
Other substrate oxidations by high-valent Fe–oxo in-
termediates that do not involve H-atom abstraction
include electrophilic attack on the aromatic ring of the
substrate by the pterin-dependent hydroxylases5and cis
dihydroxylation of an aromatic substrate by the Rieske
Significant insight into the geometric and electronic
structures of high-valent non-heme Fe–oxo complexes and
their reactivity was obtained in parallel from elegant
studies of inorganic complexes11–15(see ref 1 for a recent
review), but these studies will not be reviewed here due
to the brevity of this Account.
First Non-Heme Fe(IV)–Oxo Intermediate
A powerful approach to studying the mechanism of a
metalloenzyme-catalyzed reaction is the direct detection
of intermediates and their detailed characterization by a
combination of kinetic and spectroscopic methods. Using
this approach, one monitors changes in the geometric
and/or electronic structure of the metal center during the
reaction. This methodology had been used successfully
in the 1990s to study O2activation by the non-heme diiron
proteins methane monooxygenase and the R2 subunit of
class I ribonucleotide reductase but was only recently
applied to the mononuclear non-heme iron enzymes. The
first direct detection of an intermediate in the reaction of
a mononuclear non-heme iron enzyme with dioxygen was
reported for HPPD.16A transient absorption feature at
490 nm that forms with a second-order rate constant of
140 mM-1s-1and decays with a first-order rate constant
of 7.8 s-1was noted. More detailed spectroscopic char-
acterization of the associated intermediate has not yet
been reported. Shortly after this work, we reported the
detection and characterization of two transient states in
the reaction of taurine:RKG dioxygenase (TauD). The RKG-
dependent oxygenases are the largest and functionally
most diverse subgroup of mononuclear non-heme iron
enzymes.3They catalyze many important reactions, in-
cluding steps in the biosyntheses of antibiotics17and
collagen,18the sensing of oxygen,19–23the repair of alky-
lated DNA,24,25and the regulation of transcription by
demethylation of histones.26–28A chemical mechanism
was initially proposed more than 20 years ago by Ha-
nauske-Abel and Günzler specifically for the enzyme
prolyl-4-hydroxylase (P4H),29but its success in accom-
Scheme 1. Reactions Proposed To Be Mediated by High-Valent Fe–Oxo Intermediates
Non-Heme Fe(IV)–Oxo Intermediates Krebs et al.
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