INSIGHTS | PERSPECTIVES
Fishing for peroxidase protons
Where are the protons in heme protein catalysis?
mass produce these treatments for use in
low-income countries at a cost close to the
cost of production, with a small royalty paid
back to the pharmaceutical companies. This
is the same mechanism used to sell vaccines
in low-income countries. In some countries,
reductions in the price of HIV-1 treatments
were only achieved after long legal battles
with pharmaceutical companies. In some
cases, countries overruled company patents
on drugs and started importing generic
drugs at lower costs—so-called compulsory
licensing—which is permitted in cases of
national medical emergencies.
Creating a new funding mechanism for
poor nations is difficult in the current economic climate, but HIV has left a legacy of
structures ready to adapt to hepatitis C to
complement government and private-sec-tor efforts. UNITAID, the United Nations
agency created in 2006 to overcome market
barriers for treatments of HIV, tuberculosis,
and malaria, recently announced its first
funding for hepatitis C, with an aim of reducing treatment costs to $500 to $1000
per patient (14). It plans to scale up treatment through a multinational group of HIV
programs run by Médicins Sans Frontières,
the international medical humanitarian
organization. The Global Fund, which addresses HIV/AIDS, tuberculosis, and malaria, has funded treatment programs with
old-generation HCV drugs in several developing countries for the past 3 years.
If we can learn from the lessons of HIV/
AIDS, mass production of generics can save
millions of lives. This has been an inspiring
medical success story which need not stand
alone but can be repeated, even more rapidly, for hepatitis C. ■
REFERENCES AND NOTES
1. K. Mohd Hanafiah, J. Groeger, A. D. Flaxman, S. T. Wiersma,
Hepatology 57, 1333 (2013).
2. R. Lozano et al ., Lancet 380, 2095 (2012).
3. K. N. Ly et al., Ann. Intern. Med. 156, 271 (2012).
4. G. S. Cooke et al ., J. Viral Hepat. 20, 600 (2013).
5. A. Hill, S. Khoo, J. Fortunak, B. Simmons, N. Ford, Clin.
Infect. Dis. 58, 928 (2014).
6. Fair Pricing Coalition, Activists condemn Gilead for exorbitant price of its new hepatitis C drug; www.thebody.com/
7. N. Ford et al ., Clin. Infect. Dis. 54, 1465 (2012).
8. Medivir, 2012 Annual Report, Stockholm (2012), p. 26.
9. Bristol-Myers Squibb Company, Form 10-K: Annual report
pursuant to section 13 or 15(d) of the securities exchange
act of 1934. Fiscal year ending Dec 31, 2011, p. 9.
10. Pharmasset, Inc. Form 10-K: Annual report pursuant to
section 13 or 15(d) of the securities exchange act of 1934.
Fiscal year ending Sep 30, 2011, p. 26.
11. MSF Drug Access Team, Untangling the web of antiretroviral price reductions: 16th edition (Geneva, 2012); www.
12. B.Schwartländer, I.Grubb, J.Perriëns,Lancet 368,541
13. M. S. Sulkowski et al., N. Engl. J. Med. 370, 211 (2014).
Cytochrome c peroxidase (CcP) con- sumes hydrogen peroxide in mito- chondria, using electrons derived from reduced cytochrome c. This and a related enzyme, horseradish peroxi- dase (HRP), have played key roles in
the development of structural and mecha-
nistic biochemistry and are used in bioca-
talysis and chemiluminescent bioassays (1).
On page 193 of this issue, Casadei et al. (2)
use neutron diffraction to reveal the role and
origin of protons in heme oxidation by hydrogen peroxide,
a key step in this essential enzymatic reaction.
CcP and HRP were the
first heme enzymes for which
oxidized intermediates were
observed (1). In the textbook
mechanism for heme oxidation, protonated histidine
N-H assists O-O bond heterolysis in an Fe(III)-OOH intermediate (CcP-0), producing
CcP compound I (CcP-I) and
water. The overall course of
this reaction was established
long ago. But where are the
protons? Casadei et al. use
neutron diffraction to reveal
the positions of protons in
resting CcP and CcP-I. They
show that the iron(IV) of CcP-I is an unprotonated ferryl,
Fe(IV)=O. The results bring
new clarity to heme oxidation by hydrogen peroxide
(see the figure).
Neutron diffraction has
distinct advantages over x-ray
diffraction techniques for the structural characterization of enzymes that contain redox-active metals. Non-ionizing neutron beams
avoid the photoreduction that often plagues
structural analysis with x-rays and that also
occurs in the laser beams used for resonance
Raman spectroscopy. Laser and x-radiation
lead to ambiguities in the oxidation states
of redox-active metals such as iron or manganese. By contrast, neutrons interact only
with atomic nuclei and scatter much more
effectively from hydrogen and, especially,
deuterium atoms. Catalytic proton networks
and even deuterated hydronium ions (D3O+)
have been observed in proteins by means of
neutron diffraction (3, 4).
Efforts to understand the atomic and
electronic structure of the oxidized intermediates in the CcP catalytic cycle have been
hampered by the fact that Fe(III)/Fe(IV)
redox potentials in heme proteins lie in the
same range as those of the porphyrin ring
and those of tryptophan and tyrosine. This
“redox non-innocence” greatly increases
the complexity of these systems because it
increases the number of plausible sites of
oxidation. In HRP-I and in model porphyrin
complexes, ferryl states, Fe(IV)=O, with very
short Fe-O bond lengths have been reported
(5, 6). The distinction between Fe(IV)=O
species and their hydroxylated equivalents,
Fe(IV)-OH, has taken on considerable importance with recent evidence that cytochrome
P450 compound II is protonated and that
the basicity of ferryl oxygen strongly affects
heme protein reactivity (7).
To identify the positions of active-site
protons in CcP and CcP-I, Casadei et al.
Proton-mediated mechanism. Reaction of ferric CcP with H2O2 first gives
CcP-0, followed by O-O bond scission driven by external protonation to
afford CcP-I. Casadei et al. now report neutron diffraction data that pinpoint
the locations of the protons and elucidate the catalytic mechanism.
By John T. Groves and Nicholas C. Boaz
Department of Chemistry, Princeton University, Princeton, NJ
08544, USA. E-mail: firstname.lastname@example.org
His52 HOOH 2e, H+ –