Cytochrome: C -poly(acrylic acid) conjugates with improved peroxidase turnover number

Article abstract of DOI:10.1039/c9ob00541b

Cytochrome c-poly(acrylic acid) (cyt c-PAA) conjugates with 34-fold enchancement in peroxidase turnover number (k_cat) are reported. Cyt c-PAA conjugates were prepared by carbodiimide coupling. PAA with molecular weight (M_w) ranging from 1.8k to 250k g mol^-1 were employed, and the effect of PAA M_w on peroxiodase kinetics was assessed. The k_cat value increased with increased M_w of PAA, ranging from 0.077(±0.002) s^-1 in the absence of PAA to 2.66(±0.08) s^-1 for the conjugate of cyt c with 250k PAA. Enzymatic activity studies over pH 6-8 indicated improved activity for cyt c-PAA conjugates at neutral or slightly alkaline pH. Examination of the cyt c heme spectroscopy in the presence of H₂O₂ revealed that formation of compound III, a reactive intermediate that leads to enzyme inactivation, was supressed in cyt c-PAA conjugates. Thus, we suggest the k_cat enhancement can be attributed to acidification of the pH microenvironment and inhibition of the formation of a reactive intermediate that deactivates cyt c during the catalytic cycle.

Full text of DOI:10.1039/c9ob00541b

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DOI: 10.1039/C9OB00541B
ARTICLE
Cytochrome c-poly(acrylic acid) conjugates with improved
peroxidase turnover number
K. R. Benson,a J. Gorecki,a A. Nikiforov,a W. Tsui,a R. M. Kasi,a,b C. V. Kumar*,a,b,c
Received 00th January 20xx,
Accepted 00th January 20xx
Cytochrome c-poly(acrylic acid) (cyt c-PAA) conjugates with 34-fold enchancement in peroxidase turnover number (kcat) are
reported. Cyt c-PAA conjugates were prepared by carbodiimide coupling. PAA with molecular weight (Mw) ranging from 1.8k
to 250k g mol-1 were employed, and the effect of PAA Mw on peroxiodase kinetics was assessed. The kcat value increased
with increased Mw of PAA, ranging from 0.077(±0.002) s-1 in the absence of PAA to 2.66(±0.08) for the conjugate of cyt c
with 250k PAA. Enzymatic activity studies over pH 6-8 indicated improved activity for cyt c-PAA conjugates at neutral or
slightly alkaline pH. Examination of the cyt c heme spectroscopy in the presence of H2O2 revealed that formation of
Compound III, a reactive intermediate that leads to enzyme inactivation, was supressed in cyt c-PAA conjugates. Thus, we
suggest the kcat enhancement can be attributed to acidification of the pH microenvironment and inhibition of the formation
of a reactive intermediate that deactivates cyt c during the catalytic cycle.
DOI: 10.1039/x0xx00000x
PAA improves the thermal and chemical stability of enzymes.8,10
The effect of PAA molecular weight and polyelectrolyte features
on the catalytic activity of enzymes has not been examined. For
Introduction
Conjugation of synthetic or natural polymers to enzymes is
a proven strategy to improve the thermal and chemical stability
of enzymes.1 The use of polymeric materials, particularly
polyelectrolytes, to engineer the protein microenvironment is
emerging as an effective means to manipulate the catalytic
activity of enzyme-polymer conjugates, and can be viewed as a
type of synthetic post-translational modification.1,2 For
example, the ability of polyelectrolytes to sequester ionic
species can influence the local pH,3 which has been shown to
improve the activity of cytochrome c (cyt c),4 trypsin,5 and
chymotrypsin5 in a broad range of bulk solution pH. Non-
covalent binding of substrate or inhibitor molecules to
conjugated polymers can influence catalytic rates by enriching
local concentrations of these species, as demonstrated with
horseradish peroxidase-DNA,6 chymotrypsin-poly(quaternary
ammonium),7 and catalase-poly(acrylic acid) conjugates.8 The
polyanionic surfaces of DNA nanocages significantly improved
turnover number of several encapsulated enzymes.9 Finally,
local charge supplied by grafted polyelectrolytes can affect the
catalytic cycle of trypsin by perturbing the free energy of
reactants and intermediates.5
this reason cytochrome c-poly(acrylic acid) (cyt c-PAA)
conjugates were selected as our model system: (1) The cyt c
catalytic cycle features several cationic and anionic
intermediates,11,12 (2) the influence of polyelectrolytes on the
relative stability of these intermediates, and their mechanistic
influence enzyme turnover has not been explored and (3) the
effect of PAA molecular weight and cyt c:COOH ratio on the
kinetics of the resulting conjugates may be examined, which is
an often overlooked design consideration. With these factors in
mind, we report here cyt c-PAA conjugates with up to 34-fold
enhancement in turnover number (kcat), afforded by PAA
The above insights demonstrate that controlling the enzyme
microenvironment of polymer conjugates deserves further
research. We have previously demonstrated that conjugation to
a.Department of Chemistry, University of Connecticut, Storrs, Connecticut 06239
b.Institute of Materials Science, University of Connecticut, Storrs, Connecticut
06239
c. Department of Molecular and Cell Biology, University of Connecticut, Storrs,
Connecticut, 06239
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Figure 1. Synthesis of cyt c-PAA by EDC chemistry (top). Conjugates showed enhanced
peroxidase activity, tracked by the conversion of guaiacol (200 µM) to 3,3’-dimethoxy-4,4’-
biphenoquinone (λmax = 470 nm).
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influence on the pH microenvironment and PAA charge-
induced destabilization of wasteful reaction intermediates.
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DOI: 10.1039/C9OB00541B
Results and Discussion
Cyt c is a 12.5 kDa soluble, globular hemoprotein found in
mitochondria, where it shuttles electrons during respiration
and acts as a peroxidase during apoptosis.13 Cyt c-PAA
conjugates were formed by crosslinking PAA carboxyl groups
with the 20 primary amines of cyt c (19 lysine, 1 N-terminus)14
using carbodiimide chemistry (Figure 1). PAA (10 μM) was
activated
by
stirring
with
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC, 5-10 mM) in sodium
phosphate buffer (20 mM, pH 7.0) for 10 minutes at 25 °C. Cyt
c (10 μM) was then added, and the solution was stirred for four
hours. Conjugates were successfully produced using PAA with
average molecular weights (Mw) 1.8k, 8k, 50k, 100k, and 250k g
mol-1. Reaction by-products were routinely removed by dialysis.
Cytochrome c-poly(acrylic acid) conjugates were named as cyt
c-PAA(XXX), where the value denoted by XXX gives the
molecular weight of PAA. For example, the conjugate of cyt c
with 250k g mol-1 PAA is indicated by cyt c-PAA(250k).
Figure 2. Michealis-Menten kinetics of cyt c-PAA.
KM (M)
kcat (M s-1)
0.077(±0.002)
0.21(±0.01)
0.245(±0.005)
0.289(±0.004)
0.71(±0.016)
2.66(±0.08)
0.285(±0.004)
0.281(±0.006)
0.29(±0.01)
cyt c
cyt c-PAA(1.8k)
cyt c-PAA(8k)
cyt c-PAA(50k)
cyt c-PAA(100k)
cyt c-PAA(250k)
cyt c/PAA(50k)
cyt c/PAA(100k)
cyt c/PAA(250k)
7(±1)x10-6
7(±2)x10-6
5.1(±0.9)x10-6
4.8(±0.5)x10-6
12(±1)x10-6
38(±4)x10-6
6.8(±0.9)x10-6
10(±1)x10-6
9(±3)x10-6
Successful conjugation was validated by sodium dodecyl
sulfate polyacrylamide electrophoresis (SDS-PAGE, 12.5%
polyacrylamide), after analysis by agarose gel electrophoresis
Table 1. Michealis-Menten parameters of cyt c-PAA covalent conjugates and cyt
proved inconclusive (Figure S1). All cyt c-PAA derivatives c/PAA physical mixtures (shaded cells)
showed several bands or wide smears, indicating the broad
significantly (Welch’s t-test, p<0.05) from cyt c, but KM increased
molecular weight distribution expected from protein-polymer
nanogels.8 The disappearance of bands corresponding to free
cyt c confirmed the covalent conjugation to cyt c.
with larger molecular weight PAA, reaching up to 38(±4) μM.
The turnover number (kcat) is the rate constant for
conversion of enzyme-substrate complex to product, and was
determined using the fitted vmax. For native cyt c, kcat was
0.077(±0.002) s-1, again in good agreement with literature
reports.17 As molecular weight of PAA increased, kcat of cyt c-
PAA increased as well, ranging from 0.21(±0.01) s-1 in the case
of cyt c-PAA(1.8k) to 2.66(±0.08) s-1 for cyt c-PAA(250k). This
represented a dramatic increase in the enzymatic activity of cyt
c after conjugation to PAA, with the forward rate constant
enhanced between 3- and 34-fold.
The degree of crosslinking between cyt c and PAA was
estimated using the trinitrobenzene sulfonic acid (TNBSA) assay
to quantitate the primary amines in cyt c and cyt c-PAA (Figure
S2).15 After conjugation to PAA, the number of primary amines
of cyt c was reduced from 22.9(±0.8) to 10-13 (Table S1) for all
cyt c-PAA conjugates. No significant difference was found
among the cyt c-PAA with different Mw PAA (Welch’s t-test,
p<0.05). Thus, ~10 crosslinks were made per cyt c during the
EDC condensation.
Physical mixtures of cyt c and PAA 50k, 100k, and 250k (no
EDC) were examined next to determine if covalent conjugation
was required for activity enhancements (Figure S3). The
physical mixture of cyt c and 50k Mw PAA had a similar kcat to
the covalent adduct, which was ~4-fold greater than that of cyt
c alone. Agarose gel electrophoresis of cyt c/PAA physical
mixtures demonstrated strong non-covalent complexation of
cyt c and PAA (Figure S1A), so the enhanced activity of these
species was expected. Unlike the covalent conjugates, no
further enhancement was noted with 100k or 250k Mw PAA
physical mixtures. The full complement of Michaelis-Menten
parameters for cyt c-PAA and cyt c/PAA physical mixtures are in
Next, we assessed the effect of PAA conjugation on cyt c
peroxidase kinetics. In the catalytic cycle, hydrogen peroxide is
reduced to water, and single electron oxidation of two
equivalents of guaiacol regenerates the ferric heme state. These
two guaiacol radicals combine to form 3,3’-dimethoxy-4,4’-
biphenoquinone, which is orange in color (Figure 1).16 Oxidation
of guaiacol is the rate determining step of peroxidase activity,
so the Michaelis-Menten kinetics were examined by varying the
[guaiacol] (1-200 μM) for a fixed [H2O2] (50 mM).17 The
constants KM and vmax of cyt c and cyt c-PAA of all molecular
weights were obtained by fitting the Michaelis-Menten model
to the guaiacol-dependent initial rate data. The non-linear least
squares fits are represented by the dashed lines in Figure 2. The
KM value of cyt c for guaiacol oxidation was 7(±1) μM, orders of
magnitude smaller than that of other heme enzymes but in
good agreement with literature reports.17 The KM values of cyt
c-PAA(1.8k), cyt c-PAA(8k), and cyt c-PAA(50k) did not differ
Table 1
.
The enhancement of cyt c peroxidase activity upon binding
anionic cardiolipin has been well documented in the literature.
However, partial unfolding of cyt c and dissociation of the Fe-
Met80 bond are key to the enhanced peroxidase activity in the
presence of cardiolipin.13 A ~6-fold increase in kcat for the
2 | J. Name., 2012, 00, 1-3
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positions.24 The Soret band of native ferric cyt c haVsieawmArtaicxleimOnulimne
22,23
DOI: 10.1039/C9OB00541B
at 409 nm in the visible absorbance spectrum,
which was
observed in all cyt c-PAA except cyt c-PAA(250k). The Soret band
of cyt c-PAA(250k) shifted to 407 nm, which could be caused by
minor changes in the secondary structure.22 The Q band of
native cyt c had a maximum at 528 nm, which was maintained
in all cyt c-PAA derivatives. Finally, a weak charge transfer band
at 695 nm demonstrated the native ligation of His/Met for cyt c
and all cyt c-PAA.25 Taken together, the data indicated that the
secondary structure and heme ligation of cyt c-PAA was not
significantly changed and thus not responsible for the
enhancement in kcat.
The influence of anionic polyelectrolytes on enzyme activity
by controlling the microenvironment may provide insight into
the enhanced kinetics of cyt c-PAA. For example, encapsulation
of enzymes such as glucose oxidase in polyanionic DNA
nanocages enhanced the catalytic activity.9 Glucose oxidase and
horseradish peroxidase bound to DNA exhibit increased
cascade reaction efficiency because the phosphate backbone
maintains a favorable local pH.26 Additionally, conjugation of cyt
c to poly(methacrylic acid) was reported to change the optimum
pH of cyt c by acidifying the pH microenvironment.4 We also
studied local enrichment of guaiacol by partitioning to the PAA
Figure 3. (A) UV circular dichroism (CD) spectra of cyt c and cyt c-PAA conjugates.
Conjugate spectra are labelled with their corresponding PAA molecular weight.
(B) Soret CD spectra of cyt c and cyt c-PAA conjugates. (C) Soret (left), Q band
(center), and Met charge transfer band (right) absorbance spectra of cyt c (solid
curve) and cyt c-PAA conjugates (dashed lines).
oxidation of guaiacol has also been reported for horse cyt c phase, but guaiacol appeared to have only weak affinity for PAA
dimerized by treatment with ethanol,17 while binding to CTAB
decorated gold nanoparticles increased kcat of pyrogallol
(
Figure S5).
To test the effect of PAA on the pH activity profile, we
oxidation ~14-fold.18 Binding to functionalized carbon measured the rate of guaiacol oxidation (200 μM guaiacol, 50
nanomaterials increased vmax/KM dramatically.19 Again, these mM H2O2, 20 mM sodium phosphate) at pH 6-8. Cyt c and cyt c-
rate enhancements by others are accompanied by the breakage PAA(50k) were examined in this manner. The peroxidase
of the Met80-Fe bond and/or partial denaturation of cyt c. Thus, activity of cyt decreased as the pH was increased, while the pH
we examined cyt c-PAA to determine if the heme ligation had profile of cyt c-PAA was relatively flat (Figure 4). The Soret CD
been affected by the conjugation reaction.
and absorbance spectra did not change over this pH range,
Circular dichroism (CD) and visible absorbance spectroscopy indicating no changes to heme ligation (Figure S6). These
were employed to determine if the kcat enhancement was observations were in line previously reports of cyt c-PMMA
caused by changes to cyt c secondary structure or heme conjugates with broadened pH response.4 Thus, modulation of
ligation. Cyt c is primarily alpha helical, which was reflected in the local pH by the weakly acidic COOH groups of PAA may
the UV CD by negative peaks at 222 and 208 nm (Figure 3A).20 explain enhanced activity of cyt c-PAA.
The 208 nm band was weaker than the 222 nm band in all cases,
Examining the catalytic cycle of cyt c revealed an additional
indicative of monomeric cyt c.21 Only small changes in molar mechanism for improved catalysis by cyt c-PAA (Figure 5A). In
ellipticity were observed, demonstrating that there was little heme peroxidases, oxidation of guaiacol by occurs in three
perturbation in the protein secondary structure. Similar
changes were observed in the CD spectra of cyt c/PAA mixtures
lacking EDC, as well as in experiments where cyt c and PAA were
placed in separate cells and measured in tandem (Figure S4).
These data suggest the observed changes in the CD spectra of
cyt c-PAA could simply be artifacts caused by PAA absorbance
in the UV.
The spectra of the heme prosthetic group are highly
sensitive its ligation and binding pocket structure, which are
reflected in the Soret band (~400 nm) and Q band (~550
nm).22,23 The spectra of all cyt c-PAA samples in these regions
could be nearly overlaid on that of native cytochrome c, clearly
demonstrating that the conjugation reaction only minimally
perturbed the sensitive structure of the protein (Figure 3B-C).
The Soret CD spectra of cyt c and all cyt c-PAA derivatives
Figure 4. Variation of cyt c and cyt c-PAA(50k) rate of guaiacol oxidation with pH. Activity
showed a negative peak at 415 nm and a positive peak at 398
of cyt c decreased as pH increased, while that of cyt c-PAA increased over the same
range.
nm, indicative of native ferric cyt c with His/Met in the axial
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We hypothesized that the high negative charge density of
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PAA could suppress formation of CompoDuOnId: 1I0II.1b03y9u/Cn9fOavBo00ra5b41leB
electrostatic interactions. It is possible that PAA charge may also
stabilize the cationic Compounds I and II. However, we
examined formation of Compound III because its accumulation
can be conveniently observed spectroscopically without time-
resolved techniques under the appropriate conditions.12,27,28 To
rationalize the interaction of charged groups of PAA with heme
reactive intermediates, we estimated the Debye length in 20
mM sodium phosphate buffer at room temperature.29 Under
these conditions, the Debye length was ~1.6 nm, comparable to
the diameter of cyt c. Thus, it was reasonable to expect
electrostatic interaction between PAA charged groups and
reactive heme intermediates.
We examined the formation of Compound III by monitoring
the Q bands of cyt c and cyt c-PAA(250k) in the presence of 100
μM H2O2. (Figure 5B, Figure S7). Upon introduction of 100 μM
H2O2 to a solution of 10 μM cyt c (20 mM sodium phosphate, pH
7.0), the shoulder at 550 nm disappeared while the absorbance
at 533 nm and 563 nm increased, which indicate the
appearance of Compound III.17 These spectral changes were
complete within 30 seconds, and persisted for more than 30
minutes. Subsequent addition of guaiacol did not produce 3,3’-
dimethoxy-4,4’-biphenoquinone, which suggests that the
catalyst was deactivated. Under similar conditions, cyt c-
PAA(250k) did not show any spectral changes to indicate the
appearance of Compound III.
Next, heme bleaching studies were conducted to examine
the inactivation of cyt c and cyt c-PAA under the conditions of
the kinetic assays (20 mM sodium phosphate, pH 7.0, 20 °C). Cyt
c and cyt c-PAA(250k) were exposed to 50 mM H2O2, and the
disappearance of the Soret absorbance at 410 nm was
monitored. Interestingly, heme bleaching of cyt c-PAA(250k)
was initially more rapid that that of cyt c (Figure S8A). However,
as the reaction proceeded, the rate of cyt c-PAA(250k)
bleaching slowed relative to cyt c. After the reaction was
complete, cyt c-PAA(250k) retained 13.1(±0.9)% of its intial
Soret absorbance, while cyt c retained only 6(±1)% (Figure S8B).
In the absence of guaiacol, conjugation to PAA offered mild
protection from inactivation by heme bleaching. Inactivation by
peroxide is further inhibited by the presence of substrates
guaiacol or phenol,28 so this gain in stability can be relevant
during guaiacol oxidation.
steps.11,12 First, H2O2 is reduced and the heme iron is oxidized
from Fe(III) to Fe(IV)-oxo (Compound I). Next, the oxidation of
guaiacol produces a guaiacol radical and converts the heme to
Compound II, which is still in the Fe(IV) state. Finally, oxidation
of a second equivalent of guaiacol converts the heme back to
the native ferric state.11 Critically, Compound II can react with
H2O2 to produce Compound III, a negatively charged Fe(III)-
peroxo state. Formation of Compound III can be reversible, but
reaction of Compound III with peroxide results in enzyme
inactivation by auto-oxidation and heme bleaching.12 Even
when the enzyme is not inactivated, Compound III is a “sink” for
guaiacol oxidation, as it consumes an equivalent of hydrogen
peroxide and completes a catalytic cycle without a concomitant
oxidation of guaiacol.
Scheme 1. Schematic representation of dual influences of PAA on cyt c peroxidase activity.
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Taken together, the modulation of local pH and suppression
of wasteful intermediates could have a synergistic effect, which
result in the 34-fold enhancement in cyt c-PAA turnover number
reported here (Scheme 1). This may also help to explain the
broadened pH profile, as the ionization of PAA changes >50%
over the range studied.30 Additionally, both effects explain the
dependence of kcat on PAA Mw – increasing the Mw at a fixed cyt
c:PAA mole ratio increases the concentration of acidic groups
and magnitude of the negative charge.
We next examined if these concepts could be extended to
other heme peroxidases, such as horseradish peroxidase (HRP)
or hemoglobin (Hb). PAA conjugates of HRP and Hb were
prepared and characterized in a similar manner to cyt c-PAA,
after which Michaelis-Menten kinetics of guaiacol oxidation
were measured (Figure S9, Table S2). HRP-PAA had similar KM
and kcat to native HRP (Welch’s t-test, p<0.05). The rate constant
of inactivation of HRP by H2O2 via Compound III is at least 10-
fold smaller than that of cyt c,31,32 so the lack of improvement
was not unexpected. Hb, a typically inefficient peroxidase,33 was
improved by conjugation to PAA. kcat of Hb-PAA(50k) and Hb-
PAA(250k) were 1.1(±0.1) s-1 and 0.62(±0.04) s-1, both larger
than that of Hb, 0.43(±0.06) s-1 (Table S2). Together, the kinetics
of HRP-PAA, cyt c-PAA, and Hb-PAA indicated that the PAA could
improve the efficiency of certain enzymatic processes when the
enzymes were unstable in the presence of excess H2O2.
Acknowledgements
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This work was supported by NSF EAGEDROAI:w10a.r1d03D9M/CR9O-1B4040158471B9
and by UCONN VPR Research Excellence Award. The authors thank
Dr. Yao Lin, Jill Durso, Tawan Jamdee, Sabika Sherwani, Deaneira
Lakheram, and Caterina Riccardi for assistance.
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Conclusions
The results presented here hint at ways in which the physical
characteristics of polyelectrolytes such as PAA could be used to
tune the catalytic activity and efficiency of enzyme-polymer
conjugates. In this case, PAA destabilized a wasteful
intermediate while acidifying the local pH, thus improving the
activity of heme peroxidases, cyt c and Hb, and leading to the
34-fold increase in kcat of cyt c-PAA(250k). Significantly, by
supressing Compound III formation, PAA influenced the enzyme
mechanism. These effects could be systematically controlled by
varying the PAA Mw, allowing for precise tuning of peroxidase
activity. Additionally, these findings suggest that, with a good
understanding of the catalytic mechanism and careful polymer
selection, this concept could be extended to other enzyme-
polymer systems.
Experimental
Full experimental details are provided in the Supporting
Information.
Supporting Information
Supplementary figures and experimental methods can be found in
the Supporting Information.
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Conflicts of interest
There are no conflicts to declare.
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Cytochrome c-poly(acrylic acid) conjugates with 34-fold enhanced peroxidase activity due toacidification
of enzyme microenvironment and suppression of wasteful intermediates.