.
Angewandte
Communications
The one-electron redox potential of AaeAPO-I, [Eo(I)], is
a particularly important thermodynamic value because it is
related to the bond strength [D(O-H)] and the pKa value,
[pKa (II)] of FeIVO-H in AaeAPO-II [Eq. (3)].[5a,b] For cases in
DðOꢀHÞ ¼ n F EoðIÞ þ 2:3 R T pKaðIIÞ þ 57 ꢁ 2
ð3Þ
which Eo(I) and pKa(II) cannot be measured independently,
Equation (4) can be derived.[20] Since both Eo(I) and pKa(II)
DðOꢀHÞ ¼ nFE0cpdꢀI=cpdꢀII þ 2:3 R T pH þ 57 ꢁ 2
ð4Þ
have not been measured independently for any heme-
enzyme, the two-electron, two-proton redox potential of
AaeAPO-I measured here may be a good first approximation
of Eo(I). E0ðHRPꢀI=HRPꢀIIÞ and E0ðHRPꢀII=FerricÞ for HRP have been
measured and were found to be similar (ca. 0.95 V at
pH 6.0).[13,21] However, this result might be due to the fact
Figure 3. Calculated redox potentials, E0ðcpdꢀI=ferricÞ, as a function of
pH value for AaeAPO-I/FeIII (open squares) and CPO-I/FeIII (closed
circles) at 48C. Nernst equations for HRP-I/FeIII,[13] HOBr/Brꢀ, and
HOCl/Clꢀ are plotted in gray for comparison.
that HRP-II is not basic and exists in the FeIV O form in the
=
functional pH range. The situation is different if we consider
that AaeAPO compound II is protonated.[19] For example, if
D(O-H) is estimated to be in the range of 100 kcalmolꢀ1 [2,22]
,
Scheme 2. Nernst half-reactions for HOX and +CPorFeIV O.
=
the one-electron redox potential, E0ðcpdꢀI=cpdꢀIIÞ, would be
1.4 V vs NHE at pH 7.0, which is significantly higher than the
two-electron E0ðcpdꢀI=ferricÞ potential of 1.1 V. Accordingly, from
Equation (5), the reduction potential of AaeAPO-II
of the redox potentials parallels the reactivity of these heme
[4,14]
ꢀ
proteins. CPO-I reacts slowly with even weak C H bonds,
ꢀ
while HRP-I is barely able to oxidize C H bonds at all. By
0
0
0
III
III
2E cpdꢀI=Fe ¼ E cpdꢀI=cpdꢀII þ E cpdꢀII=Fe
ð5Þ
contrast, AaeAPO-I is highly reactive toward even very
strong C H bonds, so other active site factors may contribute
to the greater facility of C H hydroxylation than those of
CPO. Similar halide oxidation data for cytochrome P450 is
not available. However, by comparing the hydroxylation
kinetics of AaeAPO and CYP119 with similar aliphatic
substrates,[3,15] the redox properties of P450-I and AaeAPO-
I appear to lie on a similar scale.
ꢀ
À
Á
E0ðcpdꢀII=ferricÞ can be estimated to be approximately 0.8 V.
This unsymmetrical partitioning of the two redox steps may
ꢀ
ꢀ
be an important factor in facilitating homolytic C H bond
scission by heme-thiolate proteins.
In summary, the results show that chloride and bromide
ions are readily oxidized by AaeAPO-I to the corresponding
hypohalous acids. The reversibility of this oxo-transfer
reaction provides a rare opportunity to place ferryl oxo
transfers by the highly reactive heme-thiolate AaeAPO-I and
that of CPO-I on an absolute energy scale. With an estimated
bond dissociation energy for FeIVO-H in AaeAPO-II we are
able to obtain redox potentials of three redox couples
What factors contribute to the significantly higher driving
force for ferryl oxygen-atom transfer by AaeAPO-I and CPO-
I reported here as compared to that of HRP-I? The axial
ligand for AaeAPO and CPO are both cysteine thiolate
anions, while for HRP, it is a neutral, histidine nitrogen atom.
The importance of hydrogen bonding to the cysteine thiolate
of P450, CPO, and nitric oxide synthase (NOS) has been
interconnecting the resting ferric protein with its two oxidized
IV
noted.[16] According to the Nernst half-reaction (Scheme 2),
forms, +CPor-FeIV O and Fe O-H.
=
+
the driving force for the conversion of CPorFeIV O into
=
PorFeIII by oxygen-atom transfer has two contributions—the
electron affinity and the proton affinity of the ferryl species.
Although DFT calculations have indicated that the frontier
orbitals of the heme-histidine compound I are at lower energy
than the corresponding orbitals in heme-thiolate com-
pound I,[17] the strong proton affinity of a thiolate-bound
compound I may provide a large driving force resulting in
a higher net redox potential and more reactive oxidants.[5k,18]
The intrinsic basicity of the ferryl oxygen atom in
Experimental Section
Reagents: Wild-type extracellular peroxygenase of A. aegerita (iso-
form II, pI 5.6, 46 kDa) was produced in bioreactors with a soybean-
flour suspension as the growth substrate and purified as described
previously.[2,23] Kinetic experiments were performed as we have
recently described.[2] Bromination of phenol red was detected by UV/
Vis spectroscopy.[7a] At a chosen pH, 2 mL of 10 mm APO or CPO was
added to a reaction mixture containing 20 mm of phenol red (sodium
salt), 1 mm H2O2 and 10 mm NaBr.
Cys-S-FeIV O (compound II) in heme-thiolate enzymes has
=
The oxidation of ferric enzyme with NaOBr or NaOCl was
performed by stopped-flow spectroscopy with the single-mixing mode
under the diode-array or single-wavelength mode. The first syringe
was filled with enzyme in a 100 mm buffer at a chosen pH. The second
syringe was filled with the oxidant in slightly basic water solution.
been established.[19] Since Cys-S-FeIII-OH2 is the resting state,
Cys-S-FeIII-OH is also basic, thus contributing additionally to
the two-electron, two-proton oxo-transfer redox couples
determined here.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 9238 –9241