Identifying the Elusive Sites of Tyrosyl Radicals
in Cytochrome c Peroxidase: Implications for Oxidation of Substrates Bound at a Site Remote
from the Heme
posted on 2015-12-17, 02:38authored byKyle D. Miner, Thomas
D. Pfister, Parisa Hosseinzadeh, Nadime Karaduman, Lynda J. Donald, Peter C. Loewen, Yi Lu, Anabella Ivancich
The
location of the Trp radical and the catalytic function of the
[Fe(IV)O Trp191•+] intermediate
in cytochrome c peroxidase (CcP) are well-established;
however, the unambiguous identification of the site(s) for the formation
of tyrosyl radical(s) and their possible biological roles remain elusive.
We have now performed a systematic investigation of the location and
reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic
Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations
in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those
contributing primarily to the EPR spectrum of the tyrosyl radical,
recorded at 9 and 285 GHz. The EPR characterization also showed that
the heme distal-side Trp51 is involved in the intramolecular electron
transfer between Tyr71 and the heme and that formation of Tyr71• and Tyr236• is independent of the [Fe(IV)O Trp191•+] intermediate. Tyr71 is located in an optimal position to mediate
the oxidation of substrates binding at a site, more than 20 Å
from the heme, which has been reported recently in the crystal structures
of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632–1639]. The possibility of discriminating
the radical intermediates by their EPR spectra allowed us to identify
Tyr71• as the reactive species with the
guaiacol substrate. Our assignment of the surface-exposed Tyr236 as
the other radical site agrees well with previous studies based on
MNP labeling and protein cross-linking [Tsaprailis, G., and English,
A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248–255]
and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole
[Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665–11674]. Accordingly, while Tyr71 acts as a true reactive
intermediate for the oxidation of certain small substrates that bind
at a site remote from the heme, the surface-exposed Tyr236 would be
more likely related to oxidative stress signaling, as previously proposed.
Our findings reinforce the view that CcP is the monofunctional peroxidase
that most closely resembles its ancestor enzymes, the catalase-peroxidases,
in terms of the higher complexity of the peroxidase reaction [Colin,
J., et al. (2009) J. Am. Chem. Soc. 131, 8557–8563].
The strategy used to identify the elusive Tyr radical sites in CcP
may be applied to other heme enzymes containing a large number of
Tyr and Trp residues and for which Tyr (or Trp) radicals have been
proposed to be involved in their peroxidase or peroxidase-like reaction.