be used to track the temperature dependence of
the gap function in TRSB superconductors.
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Stimulating discussions with S. Kivelson, A. Huxley, and D. Agterberg;
sample characterization by K. Avers (Northwestern); and
instrument design assistance from G. Burkhard (Stanford) are greatly
appreciated. This work was supported by the U.S. Department of
Energy Office of Basic Energy Science, Division of Materials Science
and Engineering, at Stanford under contract DE-AC02-76SF00515
and at Northwestern under contract DE- FG02-05ER46248.
Construction of the Sagnac apparatus was partially funded by the
Stanford Center for Probing the Nanoscale (NSF NSEC 0425897).
E.R.S. received additional support from a Gabilan Stanford
Graduate Fellowship and the DARE Doctoral Fellowship Program.
Materials and Methods
Figs. S1 to S4
15 November 2013; accepted 21 May 2014
captures the protonation state of
ferryl heme in a peroxidase
Cecilia M. Casadei,1,2 Andrea Gumiero,3 Clive L. Metcalfe,3 Emma J. Murphy,3
Jaswir Basran,1 Maria Grazia Concilio,4 Susana C. M. Teixeira,2,5 Tobias E. Schrader,6
Alistair J. Fielding,4 Andreas Ostermann,7 Matthew P. Blakeley,2
Emma L. Raven,3 Peter C. E. Moody1*
Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl)
intermediates of the heme iron. A long-standing question has been the nature of the
iron-oxygen bond and, in particular, the protonation state. We present neutron structures
of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these
allow direct visualization of protonation states. We demonstrate that the ferryl heme is
an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the
distal histidine becomes protonated on formation of the ferryl intermediate, which has
implications for the understanding of O–O bond cleavage in heme enzymes. The structures
highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms
and visualizing protonation states in enzyme intermediates.
Aerobic organisms have evolved to use the intrinsic oxidizing power of oxygen from the atmosphere. This activation of oxy- gen is achieved by a catalytic metal center (usually iron or copper) buried within a
protein. In the case of iron, high-valent iron-oxo
(also known as ferryl) intermediates play a role
in a large number of different, and sometimes
difficult, biological oxidations catalyzed by various
heme and non-heme iron-containing enzymes.
For the heme iron enzymes, the mechanism of
oxidation involves initial formation of the iconic
compound I intermediate (1, 2). Compound I
contains an oxidized ferryl heme, plus either a
porphyrin p-cation radical or a protein radical;
reduction of compound I by one electron yields
the closely related compound II intermediate,
which contains only the ferryl heme. These com-
pound I and II intermediates are a defining fea-
ture across the heme enzyme family and appear
in a diverse group of catalytic heme enzymes
that include all the cytochrome P450s, the ni-
tric oxide synthases, and the terminal oxidases,
plus the heme dioxygenases and heme peroxi-
dases. Indeed, they are such crucial interme-
diates in so many processes—including many
involved in drug metabolism and other impor-
tant oxidations—that their structure and reactivity
has become a key question for both heme (3–7)
and non heme iron enzymes where similarly tran-
sient iron-oxo species are also employed (8, 9).
A long-standing and highly contentious question has been to clarify the bond order and protonation state of the ferryl heme. The question
has focused on whether the ferryl is formulated as an Fe(IV)=O [iron(IV)-oxo] or Fe(IV)-OH
[iron(IV)-hydroxide] species, but there are a number of reasons why previous methodologies—
none of which can measure the protonation
state directly—have failed to fully resolve the issue. To begin with, even capturing these transient intermediates in some enzymes [especially
in the P450s (10, 11)] has proved very difficult.
Early approaches to the problem used both extended x-ray absorption fine structure and resonance Raman methods [reviewed in (12)] to
examine the iron-oxygen bond as an indirect
reporter on the protonation state. These studies
indicate a short Fe-O bond length, but the data
were not totally consistent, and the photola-bility of compound I during laser excitation is
well documented (13) so that interpretation of
stretching frequencies from Raman work has not
been unambiguous either. More recently, x-ray
crystallographic methods have been employed.
These methods showed longer iron-oxygen bond
lengths, but the x-ray structures of compounds I
and II were subsequently shown to have been
affected by photoreduction of the iron and are
now considered to be unreliable. The more recent use of multiple crystals in x-ray analyses
minimizes photoreduction but cannot entirely
eliminate the problem. Moreover, hydrogen atoms
are difficult to locate in electron density maps
due to their weak scattering. Even if very high-resolution data (i.e., better than 1.2 Å) are available, hydrogen atoms can remain obscured due
to their mobility. There is also the substantive
question of whether such small changes in Fe-O
1Department of Biochemistry and Henry Wellcome Laboratories
for Structural Biology, University of Leicester, Lancaster Road,
Leicester LE1 9HN, UK. 2Institut Laue-Langevin, 71 Avenue des
Martyrs, 38000, Grenoble, France. 3Department of Chemistry,
University of Leicester, University Road, Leicester LE1 7RH, UK.
4The Photon Science Institute, The University of Manchester,
Manchester M13 9PL, UK. 5EPSAM, Keele University, Keele,
Staffordshire ST5 5BG, UK. 6Jülich Centre for Neutron Science
(JCNS), Forschungszentrum Jülich GmbH, Outstation at MLZ,
Lichtenbergstraße 1, 85747 Garching, Germany. 7Heinz
Maier-Leibnitz Zentrum (MLZ), Technische Universität München,
Lichtenbergstraße 1, D-85748 Garching, Germany.
*Corresponding author. E-mail: firstname.lastname@example.org (E.L.R.);