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Merged Heme and non-Heme Manganese Co-factors for a Dual Anti-Oxidant Surveillance in Photosynthetic Organisms Andrea Squarcina, Antonio Sorarù, Francesco Rigodanza, Mauro Carraro, Giovanna Brancatelli, Tommaso Carofiglio, Silvano Geremia, Veronique Larosa, Tomas Morosinotto, and Marcella Bonchio ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b00004 • Publication Date (Web): 09 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017
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Merged Heme and Non-Heme Manganese Co-Factors for a Dual Anti-Oxidant Surveillance in Photosynthetic Organisms Andrea Squarcina,† Antonio Sorarù,† Francesco Rigodanza,‡ Mauro Carraro,† Giovanna Brancatelli,‡ Tommaso Carofiglio,† Silvano Geremia,‡ Veronique Larosa,§ Tomas Morosinotto,§ and Marcella Bonchio*,† †
ITM-CNR and Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy. Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy. § Biology Department, University of Padova, via Bassi 58b, 35131 Padova, Italy. ‡
Dedicated to the memory of Leone Spiccia and to his research dream
ABSTRACT: coupling of a polycationic Mn(III)-porphyrin, with a dinuclear Mn2(II,II)L2 core (HL = 2-{[[di(2pyridyl)methyl] (methyl)amino]methyl}phenol), results in a dual Superoxide Dismutase (SOD) and Catalase (CAT) functional analog, Mn2L2Pn+, enabling a detoxification cascade of the superoxide anion and hydrogen peroxide into benign H2O and O2. The SOD/CAT artificial manifolds, joined in one asset, exhibit a peak catalytic performance under physiological conditions, with log kcat(O2•–) ≥ 7 and kcat(H2O2) /KM = 1890. The dual-enzyme (di-zyme) concept allows for a built-inself-protection against the irreversible bleaching of the porphyrin unit (> 75% protection), readily induced by H2O2 (200 µM, 20 equivalents, in buffer solution, pH= 7.8). We show herein that incubation of the photosynthetic green algae, Chlamydomonas reinhardtii, with the synthetic di-zyme (as low as 0.1 M), prevents H2O2 accumulation under high-light illumination conditions, thus providing anti-oxidant surveillance and photo-protection.
KEYWORDS. Artificial enzymes, manganese catalysis, superoxide dismutase, catalase, anti-oxidant catalysis, photoprotection. yield. Noteworthy, ROS-forming mechanisms are also lethal for the stability of bio-inspired materials designed for artifi4 cial photosynthesis in vitro. N
N
N Mn N
O O
O
N
+
N
N
N
O
Mn
Mn
O
N
N O
O
N
N
+ N
Mn
Mn N
N O
O
N O O
N
+
Mn N O
Mn2L2Ac+ +
N
OH
N+
N
N +N Mn N N
+
N
+
N
+
N Mn N N
HL = N
N
N
+
N
+N
+
Mn2L2P15+
N
pH=7.4
N N
+ N
Mn
N
O
N
Mn N
+
O2•-
N
O
O
N
+
Mn
N
+
H2O2
N
N
N
Mn2L2P26+
O
N N
O2
+
Oxygen is a vital component for cells, being involved in the biological energy production during respiration and photosynthesis. At the same time, living cells continuously produce highly Reactive Oxygen Species (ROS), that are toxic 1 molecules. Oxygen reduction in-vivo generates ROS, includ•– ing the superoxide anion (O2 ), hydrogen peroxide (H2O2), and the hydroxyl radical (HO•). ROS give rise to fast, barrierless, short-range and non-selective oxidation steps, being re1 sponsible for the so-called “oxidative stress”. Biological ROS surveillance stems from the combined action of key metalloenzymes, including superoxide dismutase (SOD), and cata2 lase (CAT). In particular, an effective detoxification cascade •– occurs via SOD-induced dismutation of O2 into O2 and H2O2 which is further converted by CAT into H2O and O2 again. The oxygen-rich environment of photosynthetic organisms is one crucial risk-factor for photo-oxidative stress, readily induced under intense illumination, extreme temperatures and water deficit. During photosynthesis, when the photo-induced electron-flow exceeds the transport-chain capacity, oxygen undergoes one-electron reduction forming the •– superoxide anion (O2 , the Mehler reaction), and H2O2 is eventually generated by its spontaneous or SOD-induced decay, or by a failure of the four electron water oxidation mechanism, collapsing to a bi-electronic peroxide-forming 3 process. It turns out that photo-oxidative stress is one key factor limiting plant productivity, i.e. bio-mass and food
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5+
Scheme 1. Artificial SOD/CAT di-zymes, Mn2L2P1 , 6+ + Mn2L2P2 , obtained from Mn2L2Ac by formal ligand ex-
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change with P1 or P2 synthetic porphyrins. The envisaged 6+ detoxification cascade is represented for Mn2L2P2 .
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show herein that, the di-manganese core turns out to shield the fragile heme ligand against irreversible bleaching with > 75% protection in H2O2 200 µM.
The co-delivery/co-localization of overexpressed SOD/CAT natural enzymes has been studied as an anti-ROS strategy to 5 increase tolerance. However, the SOD/CAT interplay is often plagued by their time-dependent cycles, cell-specific localiza5 tion and possible system conflicts. Building on these concepts, we have envisaged a novel, anti-ROS “domino defense” by joining SOD/CAT artificial analogs in one synthetic archi6 tecture (Scheme 1). Our approach points to a dual synthetic enzyme (di-zyme), that is conceived along bio-inspired structural guidelines, considering that: (i) SOD/CAT natural man7,8 ifolds exist both as manganese forms; (ii) Mn(SOD)s display a mono-nuclear Mn(III) active site with a nitrogen-rich 7 coordination sphere; (iii) Mn(CAT)s display a di-nuclear Mn(II)-core shaped by bridging carboxylate and oxo8 ligands. Inspection of literature data (Table S2 and S3) shows that while state-of-the-art Mn-SOD mimetics are based on mono-nuclear macrocyclic aza-complexes, only in few cases single-site Mn catalysts exhibit a dual SOD/CAT regime, in aqueous phase and with multi-turnover (TON) 6 performance. Dual SOD/CAT activity in water has been reported for Mn-salen complexes, albeit with a limited lifespan (TON = 4-17), while Mn-corroles are CAT-inactive and the Fe-analogs show a poor H2O2 conversion yield (up to 106 20%, TON= 65) and moderate SOD activity (see Table S4). The impaired activity: high SOD/low CAT efficiency, leads to H2O2 accumulation, increasing the oxidative risk and inducing an irreversible oxidative damage of the catalyst itself, as it 6 is the case of the heme-based systems.
The di-zyme assembly is conceived as an up-grade of the bio+ inspired Mn2(II)L2Bz core, (HL = 2-{[[di(2-pyridyl)methyl] 9 (methyl)amino]methyl}phenol), featuring a tetra-dentate N3O ligand set with bis-pyridyl, mono-amino and µ-oxo phenolate donors and a benzoate (Bz) bridge that connects the 9 two Mn apical positions (Scheme 1). This Mn2(II,II)-core mimics the geometrical and coordination motif of the natural CAT enzymes; however, due to a solubility issue, its CATlike activity has been successfully demonstrated only in or9 ganic solvents (i.e. CH2Cl2, MeOH, CH3CN). Modification of the benzoate linker is proposed herein with the twofold aim of (i) extending the solubility and the catalytic scope to the aqueous phase; (ii) co-assembling a SOD-mimetic unit to target a di-zyme function. As a proof-of-principle, the acetate + analogue, Mn2L2Ac (Scheme 1), has been readily isolated by 9 a modified synthetic protocol. The expected structure is confirmed in solution (ESI-MS, FT-IR, UV-vis and CV) and solid state (FT-IR and X-ray). +
X-ray analysis of Mn2L2Ac shows a highly distorted octahedral geometry for both metal centres with an overall pseudoC2 symmetry, where each Mn(II) atom is coordinated in a facial configuration by the three nitrogen atoms of the tetradentate ligand, while the phenolate and acetate ligands act as a bridge between the two metal centres (Figure 1). In par1 1 ticular, the carboxylate anion exhibits a syn–syn μ η η bridging configuration, whereby the Mn-Mn distance and the Mn−O−Mn angles of the two μ bridging phenolate residues are respectively 3.123(2)Å, 93.9(1) and 95.5(1)° (Figure 1).In + CH3CN the [Mn2L2Ac] ion is observed at m/z 777.1 by ESIMS (Figure S35) while FT-IR evidence (Figure S18) shows -1 strong absorptions at 1600 and 1576 cm that are assigned to pyridine and phenolate residues and two peaks at 1564 and
In our design, a single site Mn(III)-heme is implemented with a di-nuclear, non-heme, Mn2(II)L2 catalytic unit. Both manganese co-factors are specifically selected for a combined SOD and CAT peak activity under physiological-like conditions (Scheme 1). Accordingly, this artificial di-zyme encodes a “built-in-self-protection” against the oxidative risk. We
Table 1. Catalytic anti-oxidants featuring SOD and CAT activity in water under physiological conditions. E½/mVa vs NHE
Mn-SOD(human) Mn2-CAT (T.thermophilus) +
Mn2L2Ac
Mn2III,II/II,II
MnIII/II
IC50 (M)
log kcat(O2•–)
R0 (µM O2/s)
/
~+400
~1.3×10-9
~9.30
/
/
Mn2L2P1
5+
Mn2L2P2
6+
MnTM-4-PyP a
5+
(7) 6
/
/
/
/
/
3.1×10
+515
/
1.04×10-6
6.40
27e; 26f
0.54e; 0.52f
1245
this work
6.07
d
> 5+ + Mn2L2P1 >Mn2L2Ac (Table 1 and Table S3 for literature benchmarks), thus confirming the dominant role of the manganese heme implemented by an additional contribution 12 of the Mn2-core (Table 1). Vice-versa, the Mn2-core dictates the CAT-like activity, as similar H2O2 dismutation rates are observed for either the isolated and integrated systems (Table 1). 5+
6+
The CAT-activity of Mn2L2P1 and Mn2L2P2 (50 µM) was tested upon incubation with H2O2 (10 mM) in aqueous buff+ ers at 25.0 °C and compared with that of Mn2L2Ac . (Table 1). The Mn2-core in the conjugate maintains its CAT-like activity in borate buffer (50 mM, pH=7.8) with initial rate, R0, up to 36 µM O2/s, > 99% H2O2 conversion, turnover number, TON, -1 up to 200, and turnover frequency, TOF up to 0.73 s (Figure 2, Table 1). A similar behaviour has been also confirmed in the Krebs-Henseleit buffer (KH buffer, pH=7.4), containing a mixture of salts (sulphates, phosphates, carbonates and chlo-
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rides) and glucose, commonly used in biological solution 20 their distribution protocols (Figure S38).
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6+
(Mn2L2Ac , Mn2L2P2 , P2) in the 0.1-0.5 μM range, that showed no inhibition on algal growth. ROS production in vivo, under high-photon flux irradiation, was then evaluated using the 3,3-diaminobenzidine (DAB) colorimetric assay (Figure 4). Our results indicate that compared to the control experiment (Figure 4, No-Add), the 6+ Mn2L2P2 di-zyme provides a remarkable abatement of H2O2 accumulation (up to 60%) at a nominal concentration as low as 0.1 μM. The in-vivo screening highlights the benefit of the conjugate derivate with respect to the isolated SOD/CAT-like components: the SOD-analog, P2, turns out to be silent, while a similar H2O2 abatement is obtained with + the CAT analog, Mn2L2Ac , albeit with a five-fold concentration increase (0.5 μM, Figure 4).
+
5+
Figure 2. O2 evolution kinetics by Mn2L2Ac , Mn2L2P1 , 6+ Mn2L2P2 (50 µmol) upon incubation with H2O2 (10 mM) in BBS (50 mM pH=7.8). O2 evolution kinetics show R0 in the order 6+ 5+ + Mn2L2P2 ~Mn2L2P1 >Mn2L2Ac (Table 1). A saturation behaviour, amenable to a Michaelis-Menten treatment, has + been verified for the Mn2L2Ac precursor and for the best 6+ performing Mn2L2P2 conjugate (Figure S45 and S46). Accordingly, the CAT-like performance can be evaluated on the basis of the catalytic turnover constant, kcat(H2O2), of the Michaelis constant, KM, and of the resulting kcat(H2O2)/KM ratio (Table 1). Noteworthy the kcat(H2O2)/KM value of 1890 6+ places the Mn2L2P2 di-zyme among the top-performing artificial catalases in water (see literature benchmarks in Table S2), with a unique behaviour maintained also under saline, 2 KH buffer, conditions.
Natural SOD and CAT enzymes display diffusional rates, exceeding the rate of artificial analogs by several orders of 7, 22 magnitudes (Table 1). Their distribution in the photosynthetic cells is expected to be different, with limiting SOD at the first stage of the de-toxification chain, and with excess 23 CAT, not saturated, to avoid accumulation of toxic H2O2. In this perspective, our results are consistent with a positive effect of the di-zyme administration, whose functional advantage emerges in-vivo and can be ascribed to its unique colocalization of the SOD/CAT manifolds, as well as to the integrated heme-protection factor which shields the P2 manifold from irreversible degradation (Figure 3).
The weak point of heme-based functional systems is generally associated to the porphyrin fragility when exposed to the 21 oxidative risk. We have explored the self-protection ability 6+ 5+ of the Mn2L2P2 di-zyme compared to the MnTM-4-PyP unit upon exposure to H2O2 (50-500 µM), mimicking the oxidative stress conditions. Heme-bleaching is conveniently monitored by UV-Vis spectroscopy over time (Figure 3a). While a steady bleaching of the reference porphyrin is observed in ca. 10 minutes (Figure 3b), its coupling with the Mn2-core stops the oxidative degradation process soon after few seconds (Figure 3b ), with a porphyrin recovery in the range 85-75% based on the residual Soret absorbance (Figure S52). An anti-oxidant protection factor, p = 16, can be calcu6+ lated for Mn2L2P2 by considering the dismutation/bleaching relative rates under the conditions explored (Figure S53 and Supporting Information for calculation details). Inspection of the bleaching kinetics shows that a fast heme degradation occurs for the di-zyme soon after addition of H2O2, likely induced by a simultaneous Mn-dependent 11b self-oxidation (Inset of Figure 3b and Figure S51). The di-zyme activity was tested in vivo, by evaluating H2O2 accumulation in photosynthetic algae (Chlamydomonas reinhardtii) when exposed to high-light illumination condi-2 -1 tions (1 h with white light, 800 μmol photons m sec ). To this aim, cells were grown in tris-acetate phosphate medium (TAP) in the presence of the synthetic Mn-cofactors,
Figure 3. a) UV-Vis spectral decay recorded for MnTM-45+ PyP and b) bleaching kinetics, recorded at λ= 463 nm, for 5+ 6+ both MnTM-4-PyP and Mn2L2P2 (10 µM) upon exposure to H2O2 (200 M) in phosphate buffer (50 mM, pH=7.8); the residual porphyrin amount was estimated at λ= 463 nm by
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the ratio (At/A0). Inset shows the leveling off of the bleaching 6+ kinetics after 10 s exposure of Mn2L2P2 (8 µM) to H2O2 (150 M).
Supporting Information. Synthetic procedures, characterization details, kinetic experiments and tables with literature data. This material is available free of charge via the Internet at http://pubs.acs.org.”
ACKNOWLEDGMENT Financial support from Fondazione Cariparo (AmyCores Starting Grants2015), University of Padova (PRAT CPDA158234, and CPDA141843/14) and the ESF-COST action CMST 1205 Catalytic Routines for Small Molecule Activation (CARISMA) is gratefully acknowledged
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Figure 4. In vivo ROS detoxification of photosynthetic green algae (Chlamydomonas reinhardtii) during illumination -2 -1 (white light, 50-800 μmol photons m sec ). a) cw15 strain cells grown and incubated in the absence/presence of + 6+ Mn2L2Ac (0.5 μM), Mn2L2P2 (0.1 μM) and P2 (0.1 μM) in TAP medium (48 h) then harvested on filters (upper-row) and stained with 3,3-diaminobenzidine (DAB, 5 mM) under 1 h irradiation with high-light conditions (bottom row). b) corresponding H2O2 accumulation (DAB colorimetric test) normalized with respect to the control experiment (No Add, 100%). Statistically differences evaluated using a Student’s t test with a threshold of 0.05 (n = 9). In conclusion, the conjugation of the Mn-based SOD/CAT mimetics provides a novel di-functional anti-oxidant with an outstanding solubility in physiological conditions, peak per•– formance (logkcat(O2 ) ≥ 7; kcat(H2O2)/KM = 1890), and selfprotection (> 75% in H2O2 200 µM). These key features are instrumental in vivo, to enhance photo-protection of photosynthetic cells under oxidative stress. Photo-protection of photosynthetic organisms is crucial to implement the photosynthetic production (bio-mass and bio-synthesis) under normal or accelerated growth conditions, small molecule cofactors can thus play a key role with respect to synthetic bi24 ology tools.
AUTHOR INFORMATION Corresponding Author *
[email protected].
ASSOCIATED CONTENT
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Merged Heme and Non-Heme Manganese Co-Factors for a Dual Anti-Oxidant Surveillance in Photosynthetic Organisms Andrea Squarcina, Antonio Sorarù, Francesco Rigodanza, Mauro Carraro, Giovanna Brancatelli, Tommaso Carofiglio, Silvano Geremia, Veronique Larosa, Tomas Morosinotto and Marcella Bonchio Co-localization of SOD/CAT manganese co-factors has been achieved in water by coupling a Mn(III) cationic with a dinuclear Mn(II,II) core. This novel “di-zyme” exhibits a tandem anti-oxidant effect with peak performance (log •– kcat(O2 ) ≥ 7; kcat(H2O2) /KM = 1890) under physiological conditions, showing an unprecedented photo-protection effect in vivo within algae cells.
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