Self-Assembling Catalytic Peptide Nanomaterials Capable of Highly Efficient Peroxidase Activity

Abstract

The self-assembly of short peptides gives rise to versatile nanomaterials capable of promoting efficient catalysis. We have shown that short, seven-residue peptides bind hemin to produce functional catalytic materials which display highly efficient peroxidation activity, reaching a catalytic efficiency of 3×10⁵ m^–1 s^–1. Self-assembly is essential for catalysis as non-assembling controls show no activity. We have also observed peroxidase activity even in the absence of hemin, suggesting the potential to alter redox properties of substrates upon association with the assemblies. These results demonstrate the practical utility of self-assembled peptides in various catalytic applications and further support the evolutionary link between amyloids and modern-day enzymes.

Graphical Abstract

Supramolecular self-assembly has emerged as a powerful tool to create catalysts for numerous chemical transformations with high efficiency and selectivity.^[1] β-sheet-rich, amyloid-like peptide assemblies can facilitate hydrolytic, additive and redox transformations as well as tandem reactions.^[2] Remarkably, even fairly short and simple sequences provide ample opportunities to bind different co-factors, which drastically widens the repertoire of chemical transformations catalyzed.^[2d–g] Additionally, the dynamic nature of the assemblies provides control over catalysis using external stimuli.^[3]

Recently, we have demonstrated that assemblies formed by de novo designed peptides bind heme to promote cyclopropanation efficiently and in an enantioselective manner.^[4] This reaction requires productive association of the catalyst with multiple substrates and catalytic turnover without the loss of heme in a flat and fairly featureless system. Here, we set out to explore the potential of heme-peptide assemblies for promoting peroxidase-type activity. Natural peroxidases that utilize high valent iron species represent a very common class of enzymes capable of extremely versatile reactivity, including polymerization, hydroxylation and N, and O-dealkylations, among many others.^[5] In addition to the practical utility of peroxidases, ranging from waste water purification to polymer production and biotechnology, peroxidase activity has been extensively studied, providing many benchmarks in both enzymatic and model systems.^[5,6] Fast and easy colorimetric or fluorescent assays offer the ability to rapidly screen a lot of different molecules under multiple sets of conditions, facilitating both catalyst discovery and mechanistic studies. Finally, given the importance of metalloenzymes in nature and likely early prominence of metalloco-factors in enzymes, interactions of short peptide assemblies with heme can shed light on the emergence and evolution of early proteins.^[6g,7]

While a number of substrates can be used to test for peroxidase activity, we focused our efforts on the arguably most common ones: 3,3’,5,5’-tetramethylbenzidine (TMB), 2,2’-azinobis (3-ethylbenzthiazoline-6-sulfonate) diammonium salt (ABTS), and o-phenylenediamine (oPD; Scheme 1). Upon oxidation, all of them are converted into highly colored products. These three substrates cover a wide range of oxidation modes (a one electron process for ABTS, two one electron steps for TMB and sequential two/four electron steps for oPD),^[8] redox potentials (0.472 V for ABTS, 0.4–0.6 V for oPD and E^0’ ≈ 0.250–0.270 V for TMB, all potentials vs. AgCl/Ag)^[6e,9] and charges (oPD and TMB are neutral, ABTS is negatively charged under our experimental conditions).

Scheme 1.

Substrates used to test for peroxidase activity of hemin-binding peptide assemblies.

We have prepared a library of peptides using the previously identified heme-binding general sequence LXLHLFL (Table 1), where we have systematically explored the effect of different residues in position 2 on the reactivity. In our initial screen, we focused on TMB oxidation due to this substrate possessing the lowest redox potential. All peptides formed self-assembled structures in the presence of hemin in a 1:10 co-factor: peptide ratio to ensure full co-factor binding.^[4] Remarkably, nearly all peptide-hemin assemblies promoted oxidation, in some cases with high efficiency when compared to hemin alone (Figure S1, Supporting Information and Table 1). Interestingly, assemblies formed by LMLHLFL-hemin had the highest peroxidase activity, yet this catalyst gave only moderate yields in carbene transfer.^[4] While the limited set of peptides studied precluded us from establishing detailed structure–activity relationships we noted that hydrophobic b-branched amino acids (Ile, Val) as well as amyloidogenic (Gln) residues in position 2 promoted efficient peroxidation, while disruptions of the core structure with larger amino acids (Trp) resulted in lower activity.

Table 1.

Catalytic parameters of the peptide-hemin complexes for the oxidation of TMB with varied H₂O₂ concentrations.

Peptide	kcat [S−1]	kM (H2O2) [mM]	kcat/kM (H2O2) [M−1 S−1]
Hemin	0.179 ± 0.037	7.6 ± 1.9	24 ± 8
LHLHLFL	1.51 ± 0.21	21 ± 3	72 ± 14
LILHLFL	2.09 ± 0.30	4.1 ± 0.8	510± 124
LMLHLFL	4.41 ± 0.35	7.8 ± 0.8	565 ± 73
LGLHLFL	3.68 ± 0.46	32 ± 4	155 ± 20
LLLHLFL	0.93 ± 0.03	5.6 ± 0.2	166 ± 8
LQLHLFL	3.24 ± 0.38	13 ± 2	249 ± 48
LALHLFL	1.32 ± 0.06	4.4 ± 0.3	300 ± 25
LVLHLFL	1.96 ± 0.64	5.0 ± 2.0	392 ± 204
LHLILFL	0.442 ± 0.083	12 ± 3	35 ± 11
LFLHLFL	0.95 ± 0.12	12 ± 2	79 ± 17
LILHLWL	0.619 ± 0.025	6.4 ± 0.3	97 ± 17
LTLHLFL	0.591 ± 0.033	5.9 ± 0.4	100 ± 9

Reaction conditions: hemin and peptide were incubated for 24 h in phosphate buffer (100 mM, pH 7) before reaction. The final concentrations of hemin, the peptide and TMB were 1 μM, 10 μM, and 375 μM respectively. H₂O₂ concentrations varied between 0.125–2 mM.

Natural peroxidases are often most efficient at pH values lower than seven, so we focused our experiments on this range.^[10] Heme binding with histidine is inefficient below pH 6 due to protonation of the sidechain nitrogen, so we used this as a lower limit to avoid issues with co-factor incorporation.^[11] We explored the effect of this pH range on the assembly, hemin binding and catalytic activity of LMLHLFL-hemin, our most active peptide. Once assembled, the peptide forms welldefined fibrils (Figure S2, Supporting Information). Circular dichroism and UV/Vis data (Figures S3 and S4, Supporting Information) confirms heme binding behavior and β-sheet formation. The peptide displays greater β-sheet character with increased pH, concurrent with a small decrease in hemin extinction coefficients. Global fit of the initial rate dependence on substrate concentrations (Figure 1A) shows that TMB oxidation by LMLHLFL-hemin is consistent with a ping-pong mechanism typical for natural peroxidases.^[6h,12] Lowering the pH to 6 led to a substantial improvement of catalytic efficiency, reaching 47236±6680m^–1 s^–1 (Table 2). Encouraged by this finding, we studied the peroxidase activity of LMLHLFL-hemin assemblies using ABTS and oPD (that have high redox potential when compared to TMB) as substrates. We studied the oxidation of ABTS by H₂O₂ catalyzed by LMLHLFL-hemin by varying one substrate concentration, as well as by global fitting of the data using the ping pong model (Figure 1B, Figure S5A, Supporting Information). The catalytic efficiency demonstrated by the assemblies formed by hemin and LMLHLFL in ABTS oxidation reached 305,913±81,785m^–1 s^–1 at pH 6 (Table 2). To our knowledge, this is the highest peroxidase activity reported in any self-assembled peptide system to date, and is only an order of magnitude lower than the best examples of designed heme proteins (Table S1, Supporting Information).^[6d,13] Just like in the case of TMB, increased pH leads to a decrease of catalyt ic efficiency (Table 2, Figure S5A, Supporting Information), mostly due to increases in the K_M of the substrates. It is worth noting that the data indicates there may be some substrate inhibition of the catalyst in the oxidation of ABTS, but not in that of TMB.

Figure 1.

Initial rates of (A) TMB and (B) ABTS oxidation with H₂O₂ catalyzed by Ac-LMLHLFL-hemin (0.2 μm hemin mixed with 2 μm peptide) in 100 mm phosphate buffer at pH 6.0. Data are fit to a ping-pong kinetic model.

Table 2.

The effect of changing pH on the catalytic parameters of LMLHLFL-hemin for the oxidation of either ABTS or TMB, shown with respect to both substrate and H₂O₂. Data were fit to ping-pong kinetic model.

pH	kcat [S−1]	kM (Substrate) [μM]	kM(H2O2) [mM]	kcat/kM (Substrate) [M−1 S−1]	kcat/kM (H2O2) [M−1 S−1]
TMB
6.0	0.47 ± 0.02	9.9 ± 1.3	2.0 ± 0.2	47236±6680	235 ± 23
6.5	1.09 ± 0.08	29.6 ± 3.5	4.8 ± 0.5	36655 ± 4999	228 ± 27
7.0	0.99 ± 0.14	106 ± 21	4.4 ± 0.7	9292±2238	223 ± 48
ABTS
6.0	2.38 ± 0.19	7.8 ± 2.0	4.34 ± 0.58	305 913 ± 81 785	548 ± 85
6.5	1.85 ± 0.10	11.20 ± 0.19	2.34 ± 0.24	165179 ± 29189	791 ± 92
7.0	3.66 ± 0.17	51.70 ± 0.21	2.73 ± 0.23	70793 ± 8050	1341± 127

Reaction conditions: LMLHLFL-hemin samples were incubated for 24 h in 100 mm phosphate buffer of appropriate pH before reaction. The final concentrations of hemin and the peptide were 0.2 and 2 μm, respectively. The reaction was monitored at 652 nm for TMB or 734 nm for ABTS.

Having demonstrated the high peroxidase activity of LMLHLFL-hemin assemblies, we performed additional mechanistic studies. To probe the effects of assembly on the reactivity, we prepared the N-methylated version of the catalyst— LMLH(L^NMe)FL. The methyl group in the backbone was introduced to prevent β-sheet formation and self-assembly. CD data (Figure S3B, Supporting Information) demonstrates that while a small amount of β-sheet character remains, it is greatly reduced. While LMLH(L^NMe)FL still binds hemin (Figure S4B, Supporting Information) as indicated by a strong red shift in its UV/Vis spectrum, it shows drastically lower peroxidation activity, between 1–2 orders of magnitude less than that of LMLHLFL (Figure S5B, D, F, Supporting Information) that is not much higher than that of hemin alone.

The significantly lower K_M values observed for TMB compared to the ones for ABTS prompted us to further investigate the role of charge complementarity on the reactivity in self-assembling peptides. Since the 7-residue peptides described here cannot be easily modified to introduce charged residues without significant disruption of assembly and/or hemin binding, we turned to alternative systems to test the effect of catalyst charge on reactivity. In groundbreaking work, Fry and co-workers demonstrated that peptide amphiphiles can bind hemin to promote peroxidation.^[3b] The peptide amphiphile C₁₆-AH-LLLKKK-CO₂H was reported to have a catalytic efficiency (k_cat/K_M(H2O2)) of 32.4±4.1M^–1 S^–1 for TMB oxidation, the highest of those studied. We replaced the lysine residues with glutamates to produce a self-assembling hemin-binding peptide C₁₆-AH-LLLEEE-CO₂H, which promotes TMB oxidation with a catalytic efficiency (k_cat/K_M(H2O2)) of 238±12m⁻¹ S⁻¹, nearly an order of magnitude higher than that of the parent peptide (Figure S7B, Table S2, Supporting Information). Switching from lysine to glutamate also alters the assembly properties of the amphiphile, causing it to show strong β-sheet character at low pH values (Figure S7A, Supporting Information), but no discernible secondary structure at pH 7 or above, with heme binding demonstrated with UV/Vis spectroscopy (Figure S7B, Supporting Information). Under these conditions micelle formation was observed, consistent with the findings of Fry and co-workers (Figure S8, Supporting Information).^[3b,14] This finding suggests that charge complementarity is a key factor in oxidation catalysis and provides a straightforward path to tune catalyst properties in self-assembled systems.

Interestingly, we observed low, but still measurable, peroxidase activity with self-assembled peptides even in the absence of hemin. Given the potential evolutionary importance of co-factorless peroxidase activity in self-assembled peptide systems, we tested the heme-free oxidation abilities of LMLHLFL, the most active peptide in this study, and VHVHVQV, a peptide we have previously reported to promote oxidation of model substrates.^[2b] We found that assemblies formed by VHVHVQV promote the oxidation of oPD, TMB and ABTS in the absence of hemin (Figure S9–S14, Table S3–S5, Supporting Information). This activity is strictly co-factor independent as removal of any trace metals with Chelex did not affect the activity (Figure S12, Supporting Information). We observed a substantial substrate preference in this reaction, with oPD being favored over both ABTS and TMB. In the case of ABTS some activity was observed at pH 5.5, but not at higher pH values, and TMB oxidation was slow, so catalyst turnover was not found under our reaction monitoring conditions. While this paper was in review, Liu et al. reported the co-factor-free oxidation of TMB promoted by assemblies formed by histidine oligomer H15, however no catalyst turnover was reported (product/catalyst ratio of less than 20%).^[15] In the case of oPD oxidation promoted by VHVHVQV we observed at least two catalyst turnovers (Figure S13, Supporting Information) with maximum catalytic efficiencies reaching 0.45M⁻¹ S⁻¹ (Table 3) making it the first example of catalytic oxidation of an organic substrate promoted by peptide assemblies. Just like Liu et al.^[15] we observed &20% turnover in the case of TMB. With this caveat in mind we determined the corresponding second order rate constants to be k_cat/K_{M (TMB}) of 7M⁻¹ S⁻¹, and k_cat/K_M (_H2O2) of 1.5×10⁻² S⁻¹ (Figure S14, Supporting Information).

Table 3.

pH-Dependent kinetic parameters for the co-factor-free oxidation of oPD catalyzed by VHVHVQV assemblies.

pH	kcat (oPD) [× 10−4 S−1]	kM (oPD) [mM]	kcat/kM (oPD) [M−1 S−1]
6.0	4.7 ± 0.4	1.47 ± 0.40	0.32 ± 0.09
6.5	1.9 ± 0.1	0.42 ± 0.15	0.45 ± 0.17
7.0	1.9 ± 0.1	1.33 ± 0.26	0.14 ± 0.03

Reaction conditions: 100 μm VHVHVQV, 100 mM phosphate buffer. Samples were incubated at room temperature for 24 h before reaction.

Although the observed reaction rates in apo-VHVHVQV assemblies are significantly slower (2–3 orders of magnitude) than those of heme alone (Figure S15, Supporting Information), this is an important result showing that self-assembling peptides are capable of promoting peroxidation in the absence of any complex metalloco-factors. The mechanistic origins of this activity in fibrils are currently under investigation, but we hypothesize that the observed peroxidation activity for co-factorless assemblies is caused by the change of redox potential upon substrate association with the fibrils. Importantly, the non-assembling peptide LMLH(L^NMe)FL showed no measurable peroxidase activity (data not shown).

We have previously shown that catalytic amyloids can be easily deposited on nylon membranes to efficiently produce heterogeneous catalysts compatible with flow devices.^[2a] To determine whether heme-binding assemblies can preserve their peroxidase-like activity on a solid support, we deposited LMLHLFL-hemin assemblies on a PTFE filter and measured ABTS oxidation by passing substrate solution over it. The catalyst preserves its activity over several cycles (Figure S16A, Supporting Information), however it gradually becomes inactive (presumably due to co-factor loss) and is no longer capable of catalysis by the 12th substrate pass of the second batch of ABTS (Figure S16B, Supporting Information).

In summary, we have demonstrated that short self-assembling peptides are capable of highly efficient peroxidase-like activity. The amyloid-like fibril assemblies formed by a short seven-residue peptide, LMLHLFL, in the presence of hemin are capable of ABTS oxidation with remarkable catalytic efficiency that is to our knowledge higher than those in all reported small self-assembling peroxidases and approaches the levels shown by engineered heme proteins. Moreover, we present the first demonstration of co-factorless peroxidase-like catalysis with multiple turnovers promoted by catalytic amyloids. The catalytic fibrils can be easily deposited on solid supports for use in flow-chemistry devices, an attractive opportunity given extraordinary stability of catalytic amyloids.^[16] From a practical perspective, these findings demonstrate the ease with which complex chemical reactivity that requires high-valent iron intermediates can be supported by simple peptide assemblies and opens a path to the creation of practically useful, biocompatible, and biodegradable functional materials. From a fundamental point of view, our results further support an evolutionary link between amyloid-like assemblies and early enzymes both in the presence and absence of complex metalloco-factors.