Two oxidation sites for low redox potential substrates: A directed mutagenesis, kinetic, and crystallographic study on pleurotus eryngii versatile peroxidase

Article abstract of DOI:10.1074/jbc.M112.405548

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn²⁺ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ～70 and ～700 s^-1 mM^-1, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (～3 and ～50 s^-1 mM^-1 for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower K_m values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.

Full text of DOI:10.1074/jbc.M112.405548

Enzymology:
Two Oxidation Sites for Low Redox
Potential Substrates: A DIRECTED
MUTAGENESIS, KINETIC, AND
CRYSTALLOGRAPHIC STUDY ON
PLEUROTUS ERYNGII VERSATILE
PEROXIDASE
María Morales, María J. Mate, Antonio
Romero, María Jesús Martínez, Ángel T.
Martínez and Francisco J. Ruiz-Dueñas
J. Biol. Chem. 2012, 287:41053-41067.
doi: 10.1074/jbc.M112.405548 originally published online October 15, 2012
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 49, pp. 41053–41067, November 30, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Two Oxidation Sites for Low Redox Potential Substrates
A DIRECTED MUTAGENESIS, KINETIC, AND CRYSTALLOGRAPHIC STUDY ON PLEUROTUS
□
S
ERYNGII VERSATILE PEROXIDASE*
Received for publication, July 27, 2012, and in revised form, October 10, 2012 Published, JBC Papers in Press, October 15, 2012, DOI 10.1074/jbc.M112.405548
1
2
3
´
María Morales , María J. Mate , Antonio Romero, María Jesu´s Martínez, Angel T. Martínez ,
and Francisco J. Ruiz-Duen˜as2,4
From the Centro de Investigaciones Biolo´gicas (CIB), CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
Background: Versatile peroxidases oxidize different substrates, including low redox potential aromatics.
Results: To investigate this activity, 13 variants were characterized, and five x-ray structures obtained.
Conclusion: Phenols and dyes are oxidized in a low efficiency site in the heme channel and a high efficiency site at an exposed
tryptophan.
Significance: This study supplies new structural-functional information on versatile peroxidase, and provides variants with
improved activity on phenols.
Versatile peroxidase shares with manganese peroxidase and the substrate with Arg-43 and Pro-139 in the distal heme pocket
lignin peroxidase the ability to oxidize Mn2؉ and high redox (at the end of the heme channel) and several hydrophobic inter-
potential aromatic compounds, respectively. Moreover, it is also actions with other residues and the heme cofactor.
able to oxidize phenols (and low redox potential dyes) at two
catalytic sites, as shown by biphasic kinetics. A high efficiency
site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic
efficiencies of ϳ70 and ϳ700 s؊1 mM؊1, respectively) was local-
ized at the same exposed Trp-164 responsible for high redox
potential substrate oxidation (as shown by activity loss in the
W164S variant). The second site, characterized by low catalytic
efficiency (ϳ3 and ϳ50 s؊1 mM؊1 for 2,6-dimethoxyphenol and
p-hydroquinone, respectively) was localized at the main heme
access channel. Steady-state and transient-state kinetics for oxi-
dation of phenols and dyes at the latter site were improved when
side chains of residues forming the heme channel edge were
removed in single and multiple variants. Among them, the
E140G/K176G, E140G/P141G/K176G, and E140G/W164S/
K176G variants attained catalytic efficiencies for oxidation of
2,2ꢀ-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme
channel similar to those of the exposed tryptophan site. The
heme channel enlargement shown by x-ray diffraction of the
E140G, P141G, K176G, and E140G/K176G variants would allow
a better substrate accommodation near the heme, as revealed by
the up to 26-fold lower Km values (compared with native VP).
The resulting interactions were shown by the x-ray structure
of the E140G-guaiacol complex, which includes two H-bonds of
Ligninolytic peroxidases are enzymes of biotechnological
interest playing a central role in lignin biodegradation by white-
rot fungi (1). Three ligninolytic peroxidase families, differing in
substrate specificity, have been described and widely character-
ized (2, 3). Classical manganese peroxidase (MnP)5 (EC
1.11.1.13), first described in Phanerochaete chrysosporium,
requires Mn2ϩ to complete its catalytic cycle, and generates
Mn3ϩ that acts as a diffusible oxidizer of low redox potential
compounds (including lignin phenolic units and simple phe-
nols) (4). A new MnP type, characterized by its ability to directly
oxidize not only Mn2ϩ but also phenols and other low redox
potential compounds has been suggested in Phlebia radiata (5),
and similar genes identified in recently sequenced fungal
genomes (6–8). Unlike MnP, lignin peroxidase (LiP) (EC
1.11.1.14), first described also from P. chrysosporium, is charac-
terized by its capacity to oxidize high redox potential nonphe-
nolic aromatic substrates, including lignin model compounds
and polycyclic aromatic hydrocarbons, among others (9, 10).
Finally, versatile peroxidase (VP) (EC 1.11.1.16), thoroughly
investigated in Pleurotus eryngii, combines catalytic properties
of MnP, LiP, and generic peroxidases (low redox-potential per-
oxidases of plant and fungal origin) being able to oxidize Mn2ϩ
,
as well as phenolic and nonphenolic aromatic compounds and
dyes (11). This promiscuity is precisely what makes VP inter-
esting for different environmental and industrial applications,
including processing of plant feedstocks in the sustainable pro-
duction of fuels, chemicals, and other products (3).
* ThisworkwassupportedbytheRAPERO(BIO2008-01533), HIPOP(BIO2011-
26694), and STRUANTIVIRU (BFU2011-24615) project grants of the Spanish
Ministry of Economy and Competitiveness (MINECO) (to A. T. M., F. J. R.-D.,
and A. R., respectively) and by the PEROXICATS (KBBE-2010-4-265397)
European project (to A. T. M.).
This article contains supplemental Fig. S1.
Theatomiccoordinatesandstructurefactors(codes4FCN,4FCS,4FDQ,4FEF,and
4G05) have been deposited in the Protein Data Bank (http://wwpdb.org/).
□S
1 Supported by a Consejo Superior de Investigaciones Cient´ıficas (CSIC) I3P 5 The abbreviations used are: MnP, manganese peroxidase; ABTS, 2,2Ј-azino-
fellowship.
bis(3-ethylbenzothiazoline-6-sulfonate); CiP, Coprinopsis cinerea peroxi-
dase; DMP, 2,6-dimethoxyphenol; HQ, p-hydroquinone; kapp, apparent
second-order rate constant; kcat, catalytic constant; KD, equilibrium disso-
ciation constant; Km, Michaelis constant; kobs, pseudo first-order rate con-
stant; LiP, lignin peroxidase; PDB, Protein Data Bank; VP, versatile peroxi-
dase; VP-I/VP-II, VP compounds I and II.
2 Supported by a MINECO Ramo´n y Cajal contract.
3 To whom correspondence may be addressed. Tel.: 34-918373112; Fax:
34-915360432; E-mail: ATMartinez@cib.csic.es.
4 To whom correspondence may be addressed. Tel.: 34-918373112; Fax:
34-915360432; E-mail: FJRuiz@cib.csic.es.
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49
JOURNAL OF BIOLOGICAL CHEMISTRY 41053

Two Oxidation Sites for Low Redox Potential Substrates in VP
presents an access channel to the distal heme pocket enabling
the entrance of H2O2 for activation of the cofactor (19). In sev-
eral peroxidases, such as horseradish peroxidase (HRP) (EC
1.11.1.7) and Coprinopsis cinerea peroxidase (CiP) (EC
1.11.1.7), it is assumed that phenolic compounds are oxidized
through this channel in direct contact with the heme (20), and
the same site has been suggested for LiP oxidation of anionic
dyes (21). To determine whether the main heme access channel
is the oxidation site for phenolic substrates and dyes in VP,
directed mutagenesis was performed removing bulky and
charged residues at the channel entrance. Two of the variants
produced also included mutations at the exposed Trp-164,
which has been described as responsible for high redox poten-
tial substrate oxidation by VP (12). The steady-state and tran-
sient-state kinetics of the different variants were analyzed and
the most interesting ones were crystallized. Some of the heme-
channel mutations improved VP oxidation of phenolic com-
pounds, providing variants with a potential biotechnological
interest (22).
FIGURE 1. VP catalytic cycle proposed by Pe´rez-Boada et al. (12). Resting
state peroxidase (VP, containing Fe3ϩ) is two-electron oxidized by hydroper-
oxide, yielding compound I (VP-IA, containing Fe4ϩ-oxo and porphyrin cation
ϩ
.
radical, P ). VP-IA catalyzes one-electron oxidation of substrates in direct con-
tact with heme (e.g. Mn2ϩ) or through the formation of an alternative com-
pound I (VP-IB) containing a tryptophanyl radical, which is responsible for the
oxidation of high redox potential aromatic compounds (e.g. veratryl alcohol,
shown as VA). In both ways VP-IIA is formed. This transient state of the enzyme
bears only one oxidation equivalent (on the Fe4ϩ-oxo) and can oxidize
another substrate molecule, interacting directly with the heme group or
through the tryptophanyl radical (VP-IIB) to recover the resting state.
EXPERIMENTAL PROCEDURES
Chemicals—Catechol (purity Ն99%), dithiothreitol (purity
Ն98%), 2,6-dimethoxyphenol (DMP) (purity 99%), ferrocya-
nide (purity Ն99.99%), guaiacol (purity Ն98%), hemin (purity
Ն98%), p-hydroquinone (HQ) (purity Ն99%), isopropyl ␤-D-
thiogalactopyranoside (purity Ն99%), manganese(II) sulfate
VP combines features of the catalytic cycles of the other per- (purity Ն99.99%), and oxidized glutathione (purity Ն98%) were
oxidases mentioned above, as described by Pe´rez-Boada et al. from Sigma; H2O2, sodium tartrate (purity Ն99.5%), and urea
(12) (Fig. 1). At least seven steps can be described in this cycle, (purity Ն99.5%) were from Merck; and ABTS (purity Ͼ98%)
depending on the nature of the substrate to be oxidized. It starts was from Roche Applied Science. None of the above chemicals
when the resting enzyme (VP, containing Fe3ϩ) is two-electron was further purified.
oxidized by peroxide yielding compound I, which contains a
Heterologous Expression—Nonmutated native (wild-type)
ϩ
ferryl oxo iron (Fe4ϩ ϭ O) and a porphyrin cation radical (P ) recombinant VP and different directed variants were obtained
(VP-IA). VP-IA catalyzes one-electron substrate oxidation in by Escherichia coli expression (23). The cDNA encoding the
direct contact with heme, as in Mn2ϩ oxidation (13), or through sequence of mature isoenzyme VPL of P. eryngii (allelic variant
the formation of a tryptophanyl radical (resulting in VP-IB) (14) VPL2; GenBankTM AF007222) (24) was cloned in the pFLAG1
responsible for the oxidation of high redox potential substrates vector (International Biotechnologies Inc.) yielding pFLAG1-
(e.g. veratryl alcohol) (12). In both ways, typical compound II VPL2. E. coli DH5␣ was selected for plasmid propagation,
containing Fe4ϩ ϭ O (VP-IIA) is simultaneously formed. VP- whereas E. coli W3110 was used for native and mutated VP
IIA can also one-electron oxidize substrates directly in contact expression. The VP proteins accumulated in inclusion bodies,
with the heme or through the tryptophanyl radical characteris- and were activated in vitro and purified as indicated below.
.
tic of VP-IIB (15), restoring the resting state of the enzyme.
Site-directed Mutagenesis—Mutations were introduced by
According to the above catalytic properties, VP combines polymerase chain reaction (PCR) using the pFLAG1-VPL2
structural features of the other ligninolytic peroxidases, plasmid as template, and the QuikChangeTM kit from Strat-
although some peculiarities make it not a mere MnP/LiP hybrid agene. For each mutation, both a direct and a reverse primer
(16). In this respect, the existence of two different substrate were designed complementary to opposite strands of the same
oxidation sites is well established in VP. The Mn2ϩ oxidation DNA region, but only the direct constructions with indication
site is formed by three acidic residues at a small channel giving of the changed triplets (underlined) and the mutations intro-
access to the internal heme propionate. This catalytic site has duced (bold) are included below: (i) P76G, 5Ј-CGACACCAT-
higher plasticity than in MnP because VP is able to efficiently TGAGACTAATTTCGGCGCCAATGCTGGCATCG-3Ј; (ii)
oxidize Mn2ϩ in the absence of one of the three acidic residues F142G, 5Ј-GGACCACCTCGTGCCAGAGCCTGGTGATT-
(13). On the other hand, high redox-potential substrates are CTGTTGACTC-3Ј; (iii) K176D, 5Ј-GCCGCTGCCGACGACG-
oxidized by both VP and LiP at an exposed tryptophanyl radical, TTGACCCATCGATTCC-3Ј; (iv) K176G, 5Ј-GCCGCTGCCG-
followed by long-range electron transfer to heme (pyrrolic ACGGAGTTGACCCATCGATTCCTGG-3Ј; (v) K215Q, 5Ј-
ring-C) (12, 17). However, the two enzymes show different CCCAGGCACTGCTGACAACCAGGGAGAAGCCCAATC-
kinetic constants oxidizing substrates at this site, due to differ- TCC-3Ј; (vi) K215G, 5Ј-CCCAGGCACTGCTGACAACGGCG-
ences in the amino acid residues forming the catalytic trypto- GAGAAGCCCAATCTCC-3Ј; (vii) E140G, 5Ј-GGACCACCT-
phan environment (18). VP, like all other heme peroxidases, CGTGCCAGGACCTTTTGATTCTGTTG-3Ј; (viii) P141G,
41054 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 49•NOVEMBER 30, 2012

Two Oxidation Sites for Low Redox Potential Substrates in VP
5Ј-CCACCTCGTGCCAGAGGGTTTTGATTCTGTTGAC-
TCC-3Ј; (ix) E140G/P141G, 5Ј-CCGGACCACCTCGTGCCA-
GGCGGTTTTGATTCTGTTGACTCC-3Ј; (x) W164S, 5Ј-C-
CCGTCGAGGTTGTTTCGCTCCTGGCTTCGC-3Ј; (xi) the
double variant E140G/K176G was obtained using plasmid
pFLAG1-VPL2-E140G as template and the K176G primers;
(xii) the triple variant E140G/P141G/K176G was obtained
using plasmid pFLAG1-VPL2-E140G/P141G as template and
the K176G primers; and (xiii) the triple variant E140G/W164S/
K176G was obtained using plasmid pFLAG1-VPL2-E140G/
K176G as template and the W164S primers. The mutated genes
were sequenced using an ABI 3730 DNA Analyzer (Applied
Biosystem) to assure that only the desired mutations occurred.
PCR (50 ␮l final volume) were carried out in a PerkinElmer
Gene Amp PCR System 240 using 10 ng of template DNA, 500
␮M each dNTP, 125 ng of direct and reverse primers, 2.5 units of
Pfu Turbo polymerase (Stratagene), and the manufacturer’s
buffer. Reaction conditions were as follows: (i) a “hot start” at
95 °C for 1 min; (ii) 18 cycles at 95 °C for 50 s, 55 °C for 50 s, and
68 °C for 10 min; and (iii) a final cycle at 68 °C for 10 min.
Enzyme Production, Activation, and Purification—Native VP
and its directed variants were produced in E. coli W3110 after
transformation with the corresponding plasmids. Cells were
FIGURE 2. Chemical structures of the VP substrates used in this study.
A, guaiacol; B, DMP; C, cathecol; D, HQ; and E, ABTS.
grown for 3 h in Terrific Broth (25), induced with 1 mM isopro- Mean values Ϯ S.E. for affinity constant (Michaelis constant,
pyl ␤-D-thiogalactopyranoside, and grown for a further 4 h. The Km) and enzyme turnover (catalytic constant, kcat) were
apoenzyme accumulated in inclusion bodies, as observed by obtained by nonlinear least-squares fitting of the experimental
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, measurements to the Michaelis-Menten model. Fitting of these
and was recovered by solubilization in 50 mM Tris-HCl (pH 8.0) constants to the normalized equation: v ϭ (kcat/Km)[S]/(1 ϩ
containing 8 M urea, 1 mM EDTA, and 1 mM dithiothreitol for 30 [S]/Km) yielded the catalytic efficiency values (kcat/Km) with
min at room temperature. Subsequent in vitro folding was per- their corresponding standard errors.
formed using 0.16 M urea, 5 mM CaCl2, 20 ␮M hemin, 0.5 mM
Transient-state Kinetics—Transient-state kinetic constants
oxidized glutathione, 0.1 mM dithiothreitol, and 0.1 mg/ml of were measured at 25 °C using stopped-flow equipment (Bio-
protein in 50 mM Tris-HCl (pH 9.5) (23). Active enzyme was Logic) including a three-syringe module (SFM300) synchro-
purified by Resource-Q chromatography using a 0–0.3 M NaCl nized with a diode array detector (J&M), and Bio-Kine software.
gradient (2 ml/min, 20 min) in 10 mM sodium tartrate (pH 5.5) VP-I formation was investigated by mixing the resting enzyme
containing 1 mM CaCl2.
with increasing concentrations of H2O2 in 100 mM sodium tar-
Spectroscopic Analyses—Electronic absorption spectra were trate (pH 3.0) under pseudo first-order conditions (excess of
recorded at 25 °C using a Shimadzu UV-1800 spectrophotom- substrate) and followed at 397 nm (the isosbestic point of VP-I
eter. The concentrations of native VP and directed variants in and VP-II). To investigate VP-II formation, VP-I was first pre-
10 mM sodium tartrate (pH 5.0) were calculated from the pared by mixing 4 ␮M resting enzyme with 1 eq of H2O2 in 10
absorption at 407 nm using an extinction coefficient of 150 mM sodium tartrate (pH 5.0). After 0.6 s aging in a delay line, an
Ϫ1
Ϫ1
mM
cm (24). For spectroscopic characterization of the excess of HQ in 100 mM (final concentration) sodium tartrate
transient states in the VP catalytic cycle, 1 eq of H2O2 was added (pH 3.5) was added, and VP-II formation was followed at 416
to the resting enzyme in 10 mM sodium tartrate (pH 5.0) yield- nm (the isosbestic point of VP-II and resting enzyme). The first
ing VP-I. Addition of 1 eq of ferrocyanide to VP-I yielded VP-II. step to investigate VP-II reduction consisted of production and
Steady-state Kinetics—Oxidation of ABTS (cation radical reduction of VP-I by premixing a solution of 4 ␮M enzyme and
Ϫ1
⑀
⑀
29,300 M cmϪ1), DMP (coerulignone dimeric product 4 ␮M ferrocyanide with 1 eq of H2O2 in 10 mM sodium tartrate
436
469 55,000 MϪ1 cmϪ1), HQ (p-benzoquinone ⑀247 21,000 M
(pH 5.0). The mixture was incubated for 6 s in the delay line, and
Ϫ1
cmϪ1), catechol (o-benzoquinone ⑀392 1,456 M cmϪ1), and VP-II reduction was followed at 406 nm (the Soret maximum of
Ϫ1
guaiacol (3,3Ј-dimethoxy-4,4Ј-byphenylquinone ⑀470 26,600 resting enzyme) after mixing with different concentrations of
M
Ϫ1 cmϪ1) were estimated at pH 3.5, and that of Mn2ϩ (Mn3ϩ
-
HQ in 100 mM (final concentration) sodium tartrate (pH 3.5). In
tartrate complex ⑀238 6,500 MϪ1 cmϪ1) at pH 5.0. The chemical all cases, the final enzyme concentration was 1 ␮M. All kinetic
structures of the above substrates are shown in Fig. 2. All enzy- traces exhibited single-exponential character from which
matic activities were measured as initial velocities from linear pseudo first-order rate constants were calculated.
increments due to appearance of the reaction product, at 25 °C
Crystallization and X-ray Structure—A screening for opti-
in 100 mM sodium tartrate (of different pH values) in the pres- mal crystallization conditions by both the sitting- and hanging-
ence of 0.1 mM H2O2. Steady-state kinetic constants were cal- drop vapor diffusion methods was performed using the com-
culated from oxidation of increasing substrate concentrations. mercial Crystal Screens I and II, Index Screen, SaltRx, and
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49
JOURNAL OF BIOLOGICAL CHEMISTRY 41055

Two Oxidation Sites for Low Redox Potential Substrates in VP
TABLE 1
Data collection and refinement statistics
E140G
P141G
K176G
E140G/K176G
E140G-guaiacol
Space group
Unit cell (Å)
I41
I41
I41
I41
I41
a ϭ b ϭ 96.23
c ϭ 98.73
a ϭ b ϭ 96.58
c ϭ 98.06
a ϭ b ϭ 96.27
c ϭ 98.91
a ϭ b ϭ 96.34
c ϭ 98.94
a ϭ b ϭ 96.43
c ϭ 98.78
Resolution range (Å)
48–1.6 (1.69–1.6)a
7.7 (43.3)
68–2.0 (2.11–2.0)
11.0 (41.1)
48–1.5 (1.58–1.50)
6.2 (45.4)
48.2–1.7 (1.79–1.70)
8.3 (49.5)
69–2.35 (2.48–2.35)
18.0 (45.4)
R
merge (%)b
Multiplicity
I/␴ (I)
4.3 (4.3)
4.3 (4.3)
5.0 (4.9)
4.2 (4.2)
4.1 (4.1)
13.9 (3.2)
11.5 (3.7)
17.2 (3.3)
12.9 (3.0)
3.9 (1.6)
Completeness (%)
98.3 (97.1)
100.0 (100.0)
99.9 (99.5)
99.9 (99.7)
100.0 (99.9)
Refinement
Rc/Rfree
15.4/17.9
0.030
13.9/17.6
0.034
15.1/17.6
0.034
14.8/17.4
0.035
17.4/21.8
0.021
2.467
8.46
Bond root mean square deviation (Å)
Angle root mean square deviation (°)
Average B factor (Å2)
PDB ID
2.342
2.100
2.757
2.519
15.32
15.02
13.41
15.45
4FDQ
4FEF
4FCS
4FCN
4G05
a Data in parentheses are for the highest resolution shell.
b Rmerge ϭ ⌺hkl⌺i͉Ii (hkl) Ϫ ͗I (hkl) ͉͘/⌺hkl⌺i͉Ii (hkl)͉, where Ii(hkl) is the intensity of the ith measurement of reflection (hkl) and ͗I(hkl)͘ is the mean intensity.
c r ϭ ⌺hkl͉Fobs Ϫ Fcalc͉/⌺hkl͉Fobs ͉, where Fobs and Fcalc are the observed and calculated structure factors.
RESULTS
Additive Screen from Hampton Research. These conditions
were further refined by varying buffer type, temperature, sam-
Directed Mutagenesis at the VP Heme Channel
ple size, precipitant agent, and additives. Finally, crystals of
Several bulky residues at the main heme access channel of P.
three single (E140G, P141G, and K176G) and one double
eryngii VP (Fig. 3A) were substituted by glycines in single
(E140G/K176G) variant, as well as of the E140G-guaiacol com-
(P76G, E140G, P141G, F142G, K176G, and K215G) and multi-
plex were obtained by the sitting-drop vapor diffusion method.
ple (E140G/P141G, E140G/K176G, and E140G/P141G/
Crystals of the different variants (obtained in the absence of any
K176G) variants with the aim of facilitating the access of sub-
strates to the heme cofactor in the peroxide-activated enzyme.
In this way we attempted to make VP more similar to HRP and
CiP (Fig. 3, C and D), which are able to oxidize low redox poten-
tial substrates (e.g. simple phenols) in direct contact with the
heme. In the same way, two basic residues were replaced by
those amino acids present in P. chrysosporium LiP (Fig. 3B)
(K176D and K215Q variants) to determine the importance of
the local charge in a hypothetical substrate oxidation site at the
substrate) were soaked with the reducing substrates mentioned
above with the aim of obtaining additional enzyme-substrate
complexes.
Final crystallization conditions are provided below. The pro-
tein (10 mg/ml in 10 mM sodium tartrate, pH 5.0), or the protein
plus substrate (1:1 molar ratio) in the case of E140G-guaiacol
co-crystallization, were mixed in a 1:1 ratio (v/v) with a solution
containing 1.4 M ammonium sulfate and 100 mM sodium caco-
dylate (pH 5.0). Different additives were necessary for crystalli-
zation of each specific variant: 2% 1,3-propanediol (E140G and heme channel, as suggested for anionic dye oxidation by LiP.
K176G), 1.5% methanol (P141G), 20 mM hexamine cobalt (III)
chloride (E140G/K176G), and 2.4% inositol (E140G-guaiacol)
(final concentrations). Crystals appeared after 8–15 days at
22 °C, and were flash cooled in a cryosolution containing the
crystallization solution plus 25% glycerol.
Two more variants were produced (W164S and E140G/
W164S/K176G) lacking the catalytic Trp-164 responsible for
high redox potential substrate oxidation by VP, which is
exposed to the solvent at ϳ25 Å from the channel edge (sup-
plemental Fig. S1A).
All mutated variants were expressed in E. coli W3110, folded
in vitro, and purified. The electronic absorption spectra of the
resting and transient states (VP-I and VP-II) of these variants
exhibited the same maxima as for the native enzyme, indicating
that the mutations did not cause any substantial change in the
heme environment. The x-ray diffraction study described
below demonstrated that the mutations did not affect the over-
all protein fold, and hence the changes were basically located in
the mutated residues.
Complete data sets were collected at the beamline ID14.2 of
the ESRF (Grenoble, France). Data processing was done using
MOSFLM and reduced with SCALA, from the CCP4 package
(26). The structures were solved by molecular replacement with
MOLREP using the native VP (allelic variant VPL2) crystal
structure (PDB entry 2BOQ) as a search probe. Consecutive
cycles of refinement and manual rebuilding were done using
REFMAC (27) and COOT (28). All the refined structures were
validated using MolProbity (29). The statistics of data collec-
tion, processing, and refinement are shown in Table 1. The
atomic coordinates for the E140G, P141G, K176G, and E140G/
K176G variants and the E140G-guaiacol complex were depos-
ited in the Protein Data Bank (with accession numbers 4FDQ,
4FEF, 4FCS, 4FCN and 4G05, respectively). Although crystals
of the E140G/P141G/K176G variant could not be obtained, a
structural model for this triple variant was prepared by in silico
mutagenesis at Pro-141 of the E140G/K176G crystal structure
using the mutagenesis tool of the PyMOL Molecular Graphics
System (version 1.4.1; Schro¨dinger, LCC).
Steady-state Kinetics of Mutated Variants
The steady-state kinetic constants for oxidation of ABTS
(Fig. 2E) to a stable cation radical, and of different simple phe-
nols (Fig. 2, A–D) to the corresponding quinones, by native VP
and 13 single and multiple variants are provided below.
ABTS Oxidation—Double-hyperbolic curves were obtained
for oxidation of different concentrations of ABTS by native VP,
as shown in Fig. 4A using a semilogarithmic x axis scale. Non-
41056 JOURNAL OF BIOLOGICAL CHEMISTRY
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Two Oxidation Sites for Low Redox Potential Substrates in VP
FIGURE 3. Comparison of the heme access channel in different peroxi-
dase families. A, VP (isoenzyme VPL) from P. eryngii; B, LiP (isoenzyme H8)
from P. chrysosporium; C, HRP (isoenzyme C) from Armoracia rusticana; and
D, CiP from C. cinerea (also reported as Arthromyces ramosus “nomen nudum”
peroxidase, ARP) (PDB entries 2BOQ, 1B82, 1ATJ, and 1ARP, respectively).
Those residues forming the channel opening are indicated.
linear fitting to data for each of the curve regions enabled cal-
culation of two sets of kinetic constants (in the ␮M and mM
ranges), which revealed the existence of two oxidation sites
characterized by high and low efficiency reactions (Table 2).
Substitution of Trp-164 completely suppressed the high effi-
ciency oxidation of ABTS in the W164S and E140G/W164S/
K176G variants (Table 2) confirming the location of the high
efficiency oxidation site at this exposed tryptophan residue. By
contrast, the single mutations enlarging the heme access chan-
nel did not cause significant changes at the high efficiency site,
but affected the low efficiency oxidation of this substrate (Table
2). So, the P76G variant experienced a 3-fold decrease, whereas
the K176G and E140G variants exhibited a 4-fold increase in
their catalytic efficiency (a rebound effect was observed for the
E140G variant, with the high efficiency oxidation of ABTS
showing a simultaneous 3-fold decrease). On the other hand,
substitution of the positively charged residues at the heme
channel in the K176D and K215Q variants did not significantly
affect ABTS oxidation at the low efficiency site. This showed
that the channel charge density does not affect the oxidation of
this bulky anionic compound by VP.
FIGURE 4. Biphasic kinetics for ABTS (A), DMP (B), and HQ (C) oxidation by
native VP. Double hyperbolic curves are shown with the x axis in logarithmic
scale.
lytic Trp-164, retained the kinetic constants of the E140G/
K176G variant confirming the improvement of low efficiency
oxidation of ABTS by enlarging the heme channel; and (iv) fur-
ther channel enlargement by incorporating the P141G muta-
tion in the E140G/P141G/K176G variant did not confer an
additional improvement in ABTS oxidation compared with the
E140G/K176G variant (Table 2). When the increased catalytic
efficiency of these multiple variants at the low efficiency site
was analyzed, it was found to be basically due to the improve-
ment in affinity for ABTS (Km decrease from 1090 ␮M in native
VP to 145, 58, 56, and 41 ␮M in the E140G/P141G, E140G/
K176G, E140G/W164S/K176G, and E140G/P141G/K176G
Regarding the multiple variants: (i) the E140G/P141G double
mutation improved the affinity of the enzyme for ABTS at the
low efficiency oxidation site with respect to the already
improved E140G and P141G single variants by 2- and 3-fold,
respectively (although it reverted the kcat increase observed in
the E140G variant); (ii) the E140G/K176G double mutation
increased the catalytic efficiency of the corresponding single
variants oxidizing ABTS at the low efficiency site (up to 33-fold
compared with native VP); (iii) the triple variant E140G/
W164S/K176G, designed to avoid interferences from the cata- variants, respectively) that reached a value similar to that of the
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 41057

Two Oxidation Sites for Low Redox Potential Substrates in VP
TABLE 2
Steady-state kinetic constants (Km (␮M), kcat(s؊1), and kcat/Km (s؊1 mM؊1)) of native VP, and single and multiple variants for oxidation of ABTS at
high and low efficiency sitesa
a Reactions at 25 °C in 0.1 M tartrate (pH 3.5).
b –, not determined because of a lack of activity.
c ND, not determined because of interference with the low efficiency oxidation site. Means and 95% confidence limits.
high efficiency oxidation site in native VP (ϳ3 ␮M; Table 2). the key role of Trp-164 in the high efficiency oxidation of these
This affinity improvement, together with the characteristic phenols, as previously found for ABTS. In contrast, HQ and
Ϫ1
high kcat values of the heme catalytic site (around 200–300 s
)
DMP oxidation at the low efficiency site was maintained in both
compared with the high efficiency site (only 8 sϪ1), turned this variants. The W164S variant also showed changes in its cata-
site into an additional high efficiency site for ABTS oxidation in lytic behavior with the other two phenols assayed. A small
the better variants.
change in guaiacol Km (3.6-fold increase) and a strong effect on
Oxidation of Phenols—The steady-state kinetic constants catechol kcat (23-fold decrease), in both cases reducing the cat-
for oxidation of four different phenols (HQ, catechol, guai- alytic efficiency, were observed when this single variant was
acol, and DMP) are shown in Tables 3 and 4. Native VP compared with native VP. These results revealed that Trp-164
exhibited double hyperbolic kinetics for HQ and DMP oxi- is also involved in the high efficiency oxidation of these two
dation (Fig. 4, B and C). As in the case of ABTS, two sets of phenols, although it was not shown by the native enzyme kinet-
kinetic constants could be measured by nonlinear fitting to ics described above.
data in each of the curve regions. These sets correspond to
The enlargement of the heme channel improved the abil-
Ϫ1
Ϫ1
low (49 and 2.8 s
mM
for HQ and DMP, respectively) ity of the enzyme to oxidize the four phenols at the low
Ϫ1
Ϫ1
and high (656 and 71 s
mM
for HQ and DMP, respec- efficiency site. In particular, the P141G, K176G, E140G, and
tively) efficiency oxidation sites. By contrast, catechol and E140G/K176G mutations increased the catalytic efficiency
guaiacol oxidation apparently showed a single hyperbolic on one (HQ), two (HQ and DMP), three (HQ, DMP and
behavior, and only one set of catalytic constants, the Km guaiacol), or the four (HQ, DMP, guaiacol, and catechol)
values in the millimolar range, was obtained. This result ini- phenols, respectively (Tables 3 and 4). The observed
tially suggested the existence of a single oxidation site in improvements in the three single variants with respect to
native VP for these two phenols.
native VP were due to increases (2–10-fold) in the kcat values
When the W164S and E140G/W164S/K176G variants were (except for oxidation of guaiacol that was mainly due to the
analyzed, it was observed that they had lost the ability to oxidize 6-fold Km decrease) (Table 3). The improvement of the
HQ and DMP at the high efficiency site (Tables 3 and 4) proving E140G/K176G double variant oxidizing the four phenols was
41058 JOURNAL OF BIOLOGICAL CHEMISTRY
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Two Oxidation Sites for Low Redox Potential Substrates in VP
TABLE 3
Steady-state kinetic constants of native VP, and single variants ([Km (␮M), kcat(s؊1), and kcat/Km (s؊1 mM؊1)]) for oxidation of catechol, guaiacol,
HQ, and DMP (the last two at high and low efficiency sites)a
a Reactions at 25 °C in 0.1 M tartrate (pH 3.5).
b Not determined because saturation was not reached during reaction.
c Not determined because of a lack of activity. Means and 95% confidence limits.
a result of both the 2-fold kcat increase (with the only excep- tutions increased the catalytic efficiency toward HQ at the
tion of catechol oxidation) and the up to 4-fold decrease of low efficiency site, as described above, the double E140G/
K
m (Table 4). Although the single E140G and P141G substi- P141G variant reversed this improvement (Table 4). By con-
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49
JOURNAL OF BIOLOGICAL CHEMISTRY 41059

Two Oxidation Sites for Low Redox Potential Substrates in VP
TABLE 4
Steady-state kinetic constants of native VP, and multiple variants (Km (␮M), kcat (s؊1), and kcat/Km (s؊1 mM؊1)) for oxidation of catechol, guaiacol,
HQ, and DMP (the last two ones at high and low efficiency sites)
Reactions were at 25 °C in 0.1 M tartrate (pH 3.5).
VP
E140G/P141G
E140G/K176G
E140G/P141G/K176G
E140G/W164S/K176G
HQ (high efficiency)
a
Km
kcat
15.6 Ϯ 0.8
10.3 Ϯ 0.2
656 Ϯ 23
39.7 Ϯ 2.5
14.6 Ϯ 0.4
368 Ϯ 15
25.1 Ϯ 2.4
20.0 Ϯ 0.9
800 Ϯ 40
36.2 Ϯ 1.6
20.4 Ϯ 0.5
565 Ϯ 13
–
0
0
kcat/Km
HQ (low efficiency)
Km
kcat
716 Ϯ 25
35.1 Ϯ 0.4
49.1 Ϯ 1.0
1180 Ϯ 100
65.5 Ϯ 1.9
55.6 Ϯ 3.3
618 Ϯ 36
410 Ϯ 38
884 Ϯ 71
85.6 Ϯ 2.3
96.8 Ϯ 5.8
82.1 Ϯ 1.3
132.8 Ϯ 1.0
82.2 Ϯ 2.5
203.2 Ϯ 14.2
kcat/Km
Catechol
Km
5040 Ϯ 2001
185 Ϯ 3
10,500 Ϯ 800
105.6 Ϯ 4.0
10.1 Ϯ 0.4
2,630 Ϯ 70
185.6 Ϯ 1.3
70.7 Ϯ 1.5
4,720 Ϯ 320
135.7 Ϯ 3.7
28.8 Ϯ 1.3
34,10 Ϯ 140
164.8 Ϯ 2.8
48.4 Ϯ 1.2
kcat
k
cat/Km
36.7 Ϯ 1
Guaiacol
Km
11100 Ϯ 600
22.7 Ϯ 0.5
2.0 Ϯ 0.0
5,100 Ϯ 460
3.1 Ϯ 0.2
2,730 Ϯ 250
46.7 Ϯ 1.2
17.1 Ϯ 1.2
14,200 Ϯ 900
19.3 Ϯ 0.9
1.4 Ϯ 0.0
5,850 Ϯ 570
54.2 Ϯ 2.5
9.3 Ϯ 0.5
kcat
kcat/Km
0.6 Ϯ 0.0
DMP (high efficiency)
a
Km
kcat
78 Ϯ 8
104 Ϯ 10
4.6 Ϯ 0.1
44.6 Ϯ 3.4
189 Ϯ 14
17.3 Ϯ 0.4
91.3 Ϯ 4.5
119 Ϯ 9
–
5.6 Ϯ 0.1
71 Ϯ 6
20.4 Ϯ 0.5
171.0 Ϯ 9.7
0
0
kcat/Km
DMP (low efficiency)
Km
kcat
10500 Ϯ 400
29.8 Ϯ 0.4
2.8 Ϯ 0.1
16,000 Ϯ 800
61.0 Ϯ 1.5
3.8 Ϯ 0.1
2,970 Ϯ 130
67.0 Ϯ 0.9
22.6 Ϯ 0.8
2,380 Ϯ 200
56.6 Ϯ 1.3
23.8 Ϯ 1.6
3,330 Ϯ 260
50.2 Ϯ 1.7
15.1 Ϯ 0.7
kcat/Km
a Not determined because of a lack of activity. Means and 95% confidence limits.
trast, the E140G/P141G/K176G triple variant provided an
additional improvement with respect to the E140G/K176G
variant due to the 1.5-fold decrease of Km (Table 4).
VP-I Formation—The observed pseudo first-order rate con-
stants (k1obs) for VP-I formation (Equation 1) exhibited a linear
dependence of H2O2 concentration passing through the origin
(data not shown). Fitting k1obs versus H2O2 concentration to a
straight line yielded slope values corresponding to the apparent
second-order rate constant for VP-I formation (k1app). The sim-
ilar k1app values (ϳ3000 sϪ1 mMϪ1) obtained for native VP and
the directed variants (Table 5) indicated that mutations did not
affect formation of VP-I by H2O2.
Three additional single mutations (P76G, F142G, and
K215G) enlarging the heme channel entrance were designed,
and the resulting variants were characterized for high and low
efficiency oxidation of phenols (Table 3). The K215G variant
did not exhibit significant kinetic changes. Surprisingly, the
other two variants showed increased efficiencies for DMP
(P76G) and for DMP and HQ (F142G) at the high efficiency
oxidation site. The increase in DMP oxidation efficiency at the
high efficiency site was even more pronounced for the K176D
variant (Table 3). This is the only variant with a change in the
local charge of the main heme access that resulted in a positive
catalytic effect, although affecting a VP site (Trp-164) located at
a distant region of the enzyme.
VP-I Reduction—The kinetic traces for one-electron reduc-
tion of VP-I by HQ (Equation 2) exhibited single-exponential
character from which the pseudo first-order rate constant
(k2obs) was calculated. Plots of k2obs versus HQ concentration
were linear, corresponding to nonsaturation kinetics (Fig. 5A)
andapparentsecond-orderrateconstants(k2app)weredetermined
as the slope of a second-order plot (Table 5). The W164S mutation
did not cause any significant change in the k2app value compared
with native VP, whereas the P141G variant showed a 5-fold
decrease for this constant. The rest of mutations enlarging the
heme access channel improved the k2app values to different
extents, the E140G, E140G/W164S/K176G, and E140G/K176G
variants causing the highest increases (2.5-, 3-, and 4.3-fold,
respectively). These results confirm the important role of the
heme access channel in VP-I reduction by HQ, its enlargement
allowing better access, interaction and/or oxidation of this, and by
extension other simple phenols, reacting directly with the heme
cofactor. Moreover, the differences between the E140G/K176G
and E140G/W164S/K176G variants (the latter lacking the cata-
lytic Trp-164) suggest that both the high and low efficiency oxida-
tion sites are involved in VP-I reduction by HQ, despite reduction
by W164S had suggested that only the low efficiency site was
involved.
Transient-state Kinetics of VP-directed Variants
Kinetic constants for formation and reduction of the tran-
sient states of the VP catalytic cycle (VP-I and VP-II) were
determined using HQ as reducing substrate, which is oxidized
ꢁ
to the p-semiquinone radical (HQ ) (Equations 1–3). With this
purpose, stopped-flow spectrophotometry was performed with
native VP and the most interesting variants after the steady-
state kinetic studies (namely W164S, E140G, P141G, K176G,
E140G/K176G, E140G/P141G/K176G, and E140G/W164S/
K176G). HQ was used because its oxidation does not interfere
with the absorbance of the enzyme in the 380–450 nm range.
VP ϩ H2O2 ¡ VP-I ϩ H2O
VP-I ϩ HQ ¡ VP-II ϩ HQꢁ
(Eq. 1)
(Eq. 2)
(Eq. 3)
VP-II ϩ HQ ϩ 2Hϩ ¡ VP ϩ HQꢁ ϩ H2O
41060 JOURNAL OF BIOLOGICAL CHEMISTRY
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Two Oxidation Sites for Low Redox Potential Substrates in VP
TABLE 5
Transient-state kinetic constants of variants compared with native VP
Apparent second-order rate constants (sϪ1 mMϪ1) of VP-I formation (k1app) by H2O2, and VP-I (k2app) and VP-II reduction (k3app) by HQ. Reactions at 25 °C in 0.1 M tartrate,
pH 3.5 (pH 3 for VP-I formation), using 1 ␮M VP, final concentration, were conducted as described in the text.
b
k1app
k2app
k3appA
k3appB
VP
3,030 Ϯ 30
3,840 Ϯ 60
3,650 Ϯ 60
3,240 Ϯ 60
3,480 Ϯ 110
2,610 Ϯ 60
3,390 Ϯ 140
3,060 Ϯ 30
3,340 Ϯ 30
3,680 Ϯ 110
8,480 Ϯ 40
618 Ϯ 9
29 Ϯ 0
400 Ϯ 0
b
W164S
10 Ϯ 0
–
E140G
200 Ϯ 0
120 Ϯ 6
67 Ϯ 1
800 Ϯ 0
P141G
3,000 Ϯ 100
500 Ϯ 0
K176G
4,720 Ϯ 70
14,400 Ϯ 200
5,470 Ϯ 140
9,890 Ϯ 250
E140G/K176G
E140G/P141G/K176G
E140G/W164S/K176G
282 Ϯ 4
2,500 Ϯ 0
225 Ϯ 6
1,500 Ϯ 100
NDc
b
–
a k3appA refers to the reduction of VP-IIA, whereas k3appB refers to the reduction of VP-IIB.
b Not determined because of a lack of activity.
c Only one k3app value could be determined for this variant, which was assigned to VP-IIA reduction (k3appA). Means and 95% confidence limits.
VP-II Reduction—Pseudo first-order rate constants for one-
electron reduction of VP-II by HQ (k3obs) were also estimated.
Plots of k3obs versus HQ concentration for native VP and the
mutated variants exhibited a biphasic behavior, those of the
W164S and E140G/W164S/K176G variants (lacking the cata-
lytic Trp-164) being the only exception. With increasing sub-
strate concentration, the reduction rates were saturated or
nonsaturated at the millimolar range (Fig. 5B) and always satu-
rated in the micromolar range (Fig. 5C). The rates at the milli-
molar range were assigned to VP-IIA reduction, whereas those
at the micromolar range were attributed to VP-IIB reduction at
low and high efficiency oxidation sites, respectively (see Fig. 1).
The transient kinetic constants (k3app, k3, and KD3) of both sites
are shown in Tables 5 and 6.
VP-II reduction by those variants exhibiting saturation
kinetics can be explained by Equations 4–6, k3 and KD3 being
their first-order rate constant and equilibrium dissociation
constant, respectively.
VP-II ϩ HQ L
|
; VP-II-HQ
O
¡ VP-HQꢁ º VP ϩ HQꢁ
k3
kD3
(Eq. 4)
(Eq. 5)
(Eq. 6)
k
3obs ϭ k3͑͞1 ϩ KD3/͓HQ͔͒
K
D3 ϭ ͓VP-II͔͓HQ͔/͓VP-II-HQ͔
The apparent second-order rate constant for VP-II reduction,
k
3app (k3/KD3), was calculated by nonlinear least-squares fitting
to Equation 5 adapted as follows: k3obs ϭ (k3/KD3)[S]/(1 ϩ
[S]/KD3).
Unlike the saturation kinetics described above, the kinetics of
VP-II reduction associated to the low efficiency oxidation sites
of native VP and the K176G and E140G/K176G variants were
not saturated (Fig. 5B), so only apparent second-order rate con-
stants (k3appA) were determined.
In agreement with the steady-state results, only one set of the
transient-state kinetic constants, corresponding to the low effi-
ciency site, could be calculated for the W164S and E140G/
W164S/K176G variants (Tables 5 and 6) where the Trp-164
had been removed.
FIGURE 5. Kinetics of VP-I (A), VP-IIA (B) and VP-IIB (C) reduction by HQ by
native VP and seven mutated variants. Native VP (F) and W164S (224),
E140G (Œ), K176G (ꢂ), P141G (E), E140G/K176G (f), E140G/P141G/K176G
(ϫ), and E140G/W164S/K176G (ꢃ) variants. Reactions were at 25 °C in 0.1 M
tartrate (pH 3.5). Means and 95% confidence limits are shown.
The apparent second-order rate constant of the low effi-
ciency oxidation site (k3appA) increased in all the variants with
an enlarged heme access channel, the E140G/P141G/K176G
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49
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Two Oxidation Sites for Low Redox Potential Substrates in VP
TABLE 6
Comparison of transient-state kinetic constants for reduction of VP-II by HQ of those variants exhibiting saturation kinetics, first-order rate
constants (k3, s؊1) and equilibrium dissociation constants (KD3, ␮M) are indicated
Reactions at 25 °C in 0.1 M tartrate (pH 3.5) using 1 ␮M VP, final concentration, conducted as described in the text.
a
k3A
KD3A
k3B
11.4 Ϯ 0.1
KD3B
28.8 Ϯ 0.8
VP
NDb
NDb
c
c
W164S
11 Ϯ 1
787 Ϯ 32
206 Ϯ 8
NDb
1020 Ϯ 100
3600 Ϯ 200
1730 Ϯ 150
NDb
–
–
E140G
47.0 Ϯ 3.3
25.6 Ϯ 0.2
12.1 Ϯ 0.4
39.9 Ϯ 1.3
NDd
37.5 Ϯ 1.4
8.4 Ϯ 0.2
22.2 Ϯ 1.9
26.4 Ϯ 1.9
NDd
P141G
K176G
E140G/K176G
E140G/P141G/K176G
E140G/W164S/K176G
NDb
NDb
166 Ϯ 3
589 Ϯ 41
67 Ϯ 3
c
c
2610 Ϯ 240
–
–
a K3A and KD3A refer to the reduction of VP-IIA, whereas K3B and KD3B refer to the reduction of VP-IIB.
b Not determined because saturation was not reached during the reaction.
c Not determined because of a lack of activity.
d Only one set of k3 and KD3 constants could be determined for this variant which was assigned to VP-IIA reduction (k3A and KD3A). Means and 95% confidence limits.
variant exhibiting the highest increments (86-fold with respect nificant taking into account the variable orientation of these
to native VP) (Table 5). The improvement of this variant was residues in the eight VP crystal structures previously available
due to a significant decrease in the KD3A value (66.5 ␮M) com- in PDB (Fig. 7), none of them including mutations at this molec-
pared with the other variants exhibiting saturation kinetics ular region.
(with KD3A values in the 1–3.6 mM range) (Table 6). These vari-
The crystal structure of the E140G-guaiacol complex was
ants showed significant improvements of k3A and to a lesser solved at 2.3-Å resolution (Fig. 8). The substrate molecule is
extent of KD3A, when compared with the W164S variant (Table located at the entrance of the heme distal cavity with its aro-
6; native VP was not used as reference because it presents non- matic ring accommodated in a hydrophobic region formed by
saturation kinetics). Direct comparison of the k3A/KD3A values the heme pyrrolic ring-D, and five amino acid residues (His-47,
Ϫ1
between the W164S (11 s and 1020 ␮M, respectively) and Pro-76, Ala-77, Gly-140, and Pro-141). The methoxyl and
Ϫ1
E140G/W164S/K176G (588 s and 2610 ␮M, respectively) hydroxyl groups of guaiacol are orientated toward the internal
variants, differing only in the size of the heme access channel, cavity, hydrogen bonded to Arg-43 and Pro-139, the latter most
definitively demonstrated that a larger heme access channel probably through a water molecule (Fig. 8). A comparison of
improves VP-II reduction at the low efficiency oxidation site.
the E140G-guaiacol crystal structure with those of native VP
The k3appB also experienced an increase with the enlarge- and the other crystallized variants revealed that two conserved
ment of the heme channel, although the k3appA increments were water molecules were displaced by the substrate. Both positions
always higher (as shown for the K176G, E140G, and E140G/ are occupied by the methoxyl and hydroxyl groups of guaiacol
K176G variants) (Table 5). This phenomenon could be in the enzyme-substrate complex.
explained by interferences of VP-IIA reduction by HQ at the
DISCUSSION
heme active site on VP-IIB reduction at the catalytic Trp-164.
Overview—VP oxidizes Mn2ϩ and both high and low redox
potential aromatic compounds (11). The catalytic sites respon-
sible for oxidation of Mn2ϩ and high redox potential substrates
have been exhaustively investigated (12, 13, 15, 18). However,
not much attention has been paid to clarify how low redox
So, it was observed that the more the k3appA values improve, the
more the k3appB values increase, the P141G variant being the
only exception to this behavior.
Crystal Structures
Several attempts to crystallize the above mutated variants potential substrates are oxidized by this enzyme.
were performed in the presence and absence of reducing sub- Oxidation of low redox potential substrates, including phe-
strates (DMP, catechol, guaiacol, HQ, and ABTS) to investigate nols and small negatively charged dyes, by VP has been sug-
how the engineered mutations could affect the VP catalytic gested to occur at the exposed heme edge (22) although not
properties, as described above. Only four of the variants, in enough evidence has been provided to date. The same channel
which the ability to oxidize these substrates have been connecting the distal heme cavity with the protein surface for
improved (E140G, P141G, K176G, and E140G/K176G), could cofactor activation by exogenous H2O2 allows phenols and
be crystallized as well as the E140G variant in complex with other low redox potential compounds to gain access to the
guaiacol. Crystals obtained in the absence of substrate were heme in other members of the superfamily of microbial, fungal,
soaked with the different substrates, but additional enzyme- and plant peroxidases (30). Comparison of the heme channel
substrate complexes were not obtained.
entrance of two of these peroxidases and two ligninolytic per-
No major structural rearrangements were observed when the oxidases reveals that the channel is wider in HRP and CiP than
crystal structures of the mutated variants were compared with in LiP and VP (Fig. 3). The two ligninolytic peroxidases have
that of native VP. The main differences correspond to the size residues with bulky side chains resulting in more difficult sub-
and shape of the main channel giving access to the heme active strate access to the heme group due to steric hindrances. How-
site (Fig. 6). Differences in the orientation of the Glu-140 and ever, molecular dynamic simulations suggest that LiP heme
Glu-83 side chains were observed (the Glu-83 side chain has channel exhibits some degree of plasticity (31). Even so, the
two alternative positions with the same occupancy in the channel is too narrow in LiP to allow simple aromatic substrates
E140G/K176G crystal structure). However, they were not sig- (including phenols) to directly interact with the heme group
41062 JOURNAL OF BIOLOGICAL CHEMISTRY
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Two Oxidation Sites for Low Redox Potential Substrates in VP
FIGURE 6. Heme access channel in the crystal structures of native VP (A) and the E140G (B), P141G (C), K176G (D), and E140G/K176G (E) variants, and
the E140G/P141G/K176G model (F) mutated in silico. The different mutations are indicated and highlighted in yellow, and the solvent access surfaces are in
green. The heme group is shown as sticks and amino acid at the channel opening are both as sticks and semitransparent van der Waals spheres using
Corey-Pauling-Koltun (CPK) colors. The side chain of Glu-40 shows different orientations in native VP and the P141G and K176G variants, and Glu-83 presents
two orientations with the same occupancy in the E140G/K176G variant. The E140G variant was co-crystallized with a guaiacol molecule (shown as orange sticks
and semitransparent van der Waals spheres) that is located inside the heme channel, interacting with the enzyme as described in Fig. 8. From PDB entries: 2BOQ
(A), 4FDQ (B), 4FEF (C), 4FCS (D), and 4FCN (E).
hindering access of substrates to the heme channel. An addi-
tional aspect of the heme channel region refers to its partial
electrostatic charge. VP heme channel shares characteristics of
the other three peroxidases (HRP, CiP, and LiP) by including
one acidic (Glu-140) and two basic (Lys-176 and Lys-215)
amino acid residues. Therefore, we initially thought that the
positive net charge at this VP region could favor the oxidation
of anionic substrates (e.g. ABTS) in direct contact with the
heme group, as discussed below.
Two Sites for Low Redox Potential Substrates—The biphasic
kinetic curves obtained for different phenols and ABTS under
steady-state conditions showed the presence of two indepen-
dent catalytic sites for these substrates in native VP, character-
ized by high (Km in the micromolar range) and low (Km in the
millimolar range) specificity constants. Similar curves had been
FIGURE 7. Superimposition of residues forming the heme channel
entrance in different VP crystal structures. Selected residues from wild VP
from P. eryngii cultures (PDB 3FJW), native VP (PDB 2BOQ), and six variants
described for wild-type VP isolated from fungal cultures, and
kinetic constants for a low and a high efficiency oxidation sites
(PDB entries 2VKA, 2W23, 3FKG, 3FM1, 3FM4, and 3FMU), none of them con-
(not yet identified at that time in the VP molecular structure)
taining mutations at this region of the protein, are shown as Corey-Pauling-
had been provided (11, 33).
Koltun (CPK) sticks, whereas the Glu-83 and Glu-140 residues in the P141G
(PDB 4FEF), K176G (PDB 4FCS), and E140G/K176G (PDB 4FCN) variants are
shown as orange, magenta, and yellow sticks, respectively. The heme cofactor
is shown at the bottom of the channel (CPK sticks). All the variants were
Taking the above considerations together, size and local
charge of the VP main heme access channel were analyzed in
expressed in E. coli.
depth to elucidate its eventual role in oxidation of low redox
potential substrates. VP-directed variants were prepared by
substituting amino acid residues with bulky side chains by gly-
cines, and by replacing basic residues to reduce or reverse the
positive partial charge. Moreover, the above mutations were
combined with the removal of Trp-164, which has been
(32), but not so in VP that has a wider channel. Moreover, cer-
tain mobility of the side chains located at the heme channel
opening in VP is suggested by the superimposition of crystal
structures available at PDB, somehow emulating the snapshots
of a molecular dynamics simulation (Fig. 7). These crystal struc- reported as responsible for oxidation of high redox potential
tures also reveal that Glu-140 could play a role facilitating or substrates (12) and also ABTS (18) by P. eryngii VP.
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49
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Two Oxidation Sites for Low Redox Potential Substrates in VP
dation of DMP and HQ, and impaired catechol and guaiacol
oxidation by the W164S variant confirm this fact. The steady-
and transient-state kinetic constants for oxidation of phenols
by native VP are similar to those reported for LiP oxidation of
phenols (34) (namely Km/KD3B values in the micromolar range,
and kcat/k3B values of 5–20 sϪ1). This suggests that the homol-
ogous tryptophan in P. chrysosporium LiP (Trp-171), being
responsible for high redox potential substrate oxidation (21),
would be also involved in oxidation of phenols by this enzyme.
However, unlike VP, LiP is rapidly inactivated during oxidation
of phenolic substrates (35, 36), this being the reason why phe-
nols are not considered among its natural substrates.
Low Efficiency Oxidation at the ␦-Heme Edge—Exhaustive
analysis of variants mutated at the heme channel has confirmed
this site as involved in the low efficiency oxidation of phenols
and ABTS by VP. The affinity constants of native VP at this site
were near (HQ) or in the millimolar (ABTS and other phenols)
range. These values are similar to the Km and KD values reported
for generic peroxidases oxidizing the same type of substrates at the
␦-meso-position of the porphyrin macrocycle (the so-called
␦-heme edge) (supplemental Fig. S1B) (37–39). These data suggest
bothsimilarbindingofthesesubstratesbygenericperoxidasesand
VP, despite the aforementioned differences in their heme access
channels, and minimal interferences by contaminants present at
substrate concentrations in the millimolar range (substrates were
not 100% pure, as described under “Experimental Procedures”).
Relevant changes in the affinity and catalytic constants of the low
efficiency sitewereobservedwhentheVPchannelwas enlarged by
selected amino acid substitutions by glycines. The effect became
more evident when multiple substitutions were included in double
(E140G/K176G) and triple (E140G/P141G/K176G) variants. The
catalytic properties of the E140G/W164S/K176G variant (lacking
Trp-164) left no doubts about the existence of this second site for
oxidation of low redox potential substrates by VP. These results
revealed a general tendency of the enzyme (depending on each
variant and substrate) to improve its catalytic efficiency at the low
efficiency site when an enlargement of the heme channel was
produced.
The exposed ␦-heme edge has been also described as the site
for oxidation of a negatively charged difluoroazo dye by LiP,
and this reaction has been influenced by a charge neutralization
mutation in the “classic” heme edge access channel (21). In
contrast, we observed that changes in the local charge of the
entrance to the heme channel in VP did not have any relevant
effect on ABTS oxidation. However, this could be due to the
large size of the ABTS molecule that could interact with oppo-
sitely charged residues at some distance from the entrance of
the heme access channel.
FIGURE 8. Guaiacol at the heme access channel of the E140G-guaiacol
complex. A, 2Fo Ϫ Fc electron density map, contoured at the 1.1 ␴ level, of the
guaiacol molecule within the E140G-guaiacol complex. The guaiacol mole-
cule was located near the position of Arg-43, Pro-139, and two water mole-
cules (W145 and W172), and the heme cofactor (waters are shown as red
spheres; and therestasCorey-Pauling-Koltun(CPK)colored sticks). B, LIGPLOT
(41)diagramofguaiacolinteractionswiththeE140Gvariant. HBPLUS(42)was
used to calculate hydrogen bonds and hydrophobic contacts (the latter are
interpreted by following the spokes protruding from a ligand atom toward a
protein residue, which is shown as an arc). Guaiacol bonds are depicted in
purple line and those of the protein in brown line; and hydrogen bonds, and
their lengths, are shown in green.
VP-I/VP-II Reduction at Trp-164 or ␦-Heme Edge—As dis-
cussed above, the steady-state kinetics of native VP and its
mutated variants allowed us to identify two independent sites,
High Efficiency Oxidation at Trp-164—After H2O2 activa- characterized by their different kinetic properties (a combina-
tion, both VP transient states present a protein radical at the tion of high affinity and low catalytic constants for the high
Trp-164 residue (15), which is exposed to the solvent at ϳ25 Å efficiency site at Trp-164; and low affinity and high catalytic
from the channel edge (see supplemental Fig. S1A). According constants for the low efficiency site at the heme edge). These
to the results presented herein, the substrate specificity of this differences were also used for estimating two sets of transient-
catalytic site can be extended to simple phenols, such as DMP, state kinetic constants, revealing that both VP-I and VP-II are
HQ, catechol, and guaiacol. Suppression of high efficiency oxi- reduced by phenols reacting in the two catalytic sites. So, the VP
41064 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 49•NOVEMBER 30, 2012

Two Oxidation Sites for Low Redox Potential Substrates in VP
action after access of the substrate in native VP. In conse-
quence, a Glu-140 “open” orientation seems necessary to allow
the substrate to enter and optimally interact with heme and
surrounding amino acid residues in native VP, as observed in
the E140G-guaiacol complex.
When analyzed, the guaiacol binding mode in the E140G
variant showed common features with crystal structures of
microbial, fungal, and plant peroxidase (cytochrome c peroxi-
dase, CiP, HRP, and ascorbate peroxidase) complexes with iso-
niazid, ferulic, and hydroxamic acids (reviewed in Ref. 19). The
most similar structures to the E140G-guaiacol complex were
those of HRP-ferulic acid and HRP-cyanide-ferulic acid com-
plexes (40). These structures share the same orientation of the
aromatic ring and its substituents in the ferulic acid and guaia-
col complexes. Ferulic acid orientation in the HRP-cyanide-
ferulic acid complex was suggested to be the same as it would be
in a complex with compounds I or II (cyanide parallels the
FIGURE 9. Updated VP catalytic cycle including phenols as VP-I and VP-II
structural effect of the ferryl oxygen in these compounds) (40).
In consequence, the same orientation of the guaiacol molecule
observed in the crystallized E140G-guaicol complex is expected
to be maintained in the two other states of VP catalytic cycle. In
this context, conserved residues at the heme distal pocket are
directly involved in enzyme-substrate interaction, including a
proline (VP Pro-139) backbone carbonyl and the guanidinium
side chain of the distal arginine (VP Arg-43), which are hydro-
gen bonded to the guaiacol hydroxyl and methoxyl oxygens,
respectively, in the VP-phenol complex. Additionally, two dis-
tal water molecules seem to be important in the stabilization of
the E140G-guaiacol and HRP-ferulic acid complexes, mediat-
ing the substrate interaction with the heme group.
reducingsubstrates. Athighconcentrations(millimolarrange)phenolsgain
access to the heme active site and are oxidized in direct contact with the
␦-position of the porphyrin macrocycle by VP-IA [Fe4ϩ ϭ O P ] and VP-IIA
ꢁ
[Fe4ϩ ϭ O], but when present at low concentrations (micromolar range) they
are preferentially oxidized at a protein radical centered on Trp-164 exposed
to the solvent by VP-IB [Fe4ϩ ϭ O Trp ] and VP-IIB [Fe Trp ]. The rest of the
3ϩ
ꢁ
ꢁ
catalytic cycle is as previously described by Pe´rez-Boada et al. (12) (Fig. 1).
catalytic cycle initially proposed by Ruiz-Duen˜as et al. (24), and
then extended by Pe´rez-Boada et al. (12) (Fig. 1), can be now
completed as shown in Fig. 9. According to this scheme, phe-
nols at micromolar concentrations are oxidized by the Trp-164
radical in both VP-IB and VP-IIB, as previously demonstrated
for high redox potential substrates (15). By contrast, phenols at
millimolar concentrations are oxidized in direct contact with
the heme cofactor by VP-IA and VP-IIA. According to our stud-
ies, phenols gain access to the ␦-heme edge through the main
channel, whereas Mn2ϩ, which is also oxidized directly by the
heme, accesses to the internal propionate through a small sec-
ondary channel formed by the three acidic residues participat-
ing in its coordination (13).
Conclusions—Previous studies demonstrated how Mn2ϩ and
high redox potential substrates are oxidized by VP, but left aside
the oxidation of low redox potential phenols. Here two catalytic
sites responsible for low redox potential substrate oxidation
were identified. The high efficiency site corresponds to the
same Trp-164 involved in high redox potential substrate oxida-
tion. We describe for the first time in VP (and related LiP that
shares the exposed catalytic tryptophan) how the substrate
range of this site extends to simple phenols. In parallel, a low
efficiency site, involved in oxidation of the same phenols, was
localized at the entrance of the VP heme distal pocket, being
absent from P. chrysosporium LiP and MnP. This catalytic site
has been exhaustively characterized, and the structural determi-
nants responsible for the enzyme-substrate interaction have been
identified in the molecular structure of the E140G variant after
co-crystallization with guaiacol. With the results herein presented,
we conclude the description of the different catalytic sites present
in VP, as a model ligninolytic peroxidase, and we provide addi-
tional clues on the possible catalytic mechanisms/sites for oxida-
tion of similar substrates in structurally related peroxidases.
Structural Evidence for Heme Channel Oxidation Site—Op-
timized enlargement of the VP heme channel yielded a variant
(E140G) that could be co-crystallized with a guaiacol molecule
interacting with the pyrrolic ring-D of heme. This is the second
in vivo phenolic substrate, ferulic acid was the first one (40), ever
crystallized at the active site of a peroxidase. The weakness of the
binding interactions for a stable enzyme-substrate complex is the
most plausible reason for the absence of crystallographic informa-
tion for a peroxidase with a phenolic substrate, in agreement with
the high dissociation constants determined for some of them (38,
39). Our results with the VP-guaiacol complex seem to be consist-
ent with this hypothesis. Only the E140G variant, showing a sig-
nificant affinity improvement for guaiacol, yielded crystals with
this compound located at the exposed ␦-heme edge.
On the other hand, we have demonstrated the VP catalytic
improvement associated to the Glu-140 removal. As shown in
Fig. 7, the Glu-140 side chain occupies different positions in
different VP crystal structures, in agreement with the absence
of steric restrictions for free side chain mobility. This suggests
that Glu-140 could temporally occlude the substrate access to
the heme active site or destabilize the enzyme-substrate inter-
Acknowledgments—We thank Dr. K. Piontek (University of Freiburg,
Germany) for making available in PDB several unpublished VP crys-
tal structures. We are grateful to the scientists of beamline ID23-2
(ESRF, Grenoble, France) for help during data collections. We also
acknowledge SOLEIL for provision of synchrotron radiation facilities
(proposal ID “20100576”).
NOVEMBER 30, 2012•VOLUME 287•NUMBER 49
JOURNAL OF BIOLOGICAL CHEMISTRY 41065

Two Oxidation Sites for Low Redox Potential Substrates in VP
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