Superoxide radical anion scavenging and dismutation by some Cu2+ and Mn2+ complexes: A pulse radiolysis study

Radiation Physics and Chemistry 139 (2017) 74–82

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Superoxide radical anion scavenging and dismutation by some Cu2+ and Mn2+ complexes: A pulse radiolysis study

MARK

Ravi Joshi Radiation & Photochemistry Division, Chemistry Group, Bhabha Atomic Research Center, Mumbai 400085, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Cu2+ Mn2+ Natural ligands Pulse radiolysis SOD mimic

Copper (Cu) and manganese (Mn) ions are catalytic centers, in complexed form, in scavenging and dismutation process of superoxide radicals anion (O2.−) by superoxide dismutase enzyme. In the present work, fast reaction kinetics and mechanism of scavenging and dismutation of O2.− by Cu2+, Mn2+ and their complexes formed with some natural ligands have been studied using pulse radiolysis technique. Catechol, gentisic acid, tetrahydroxyquinone, tyrosine, tryptophan, embelin and bilirubin have been used as low molecular weight natural ligands for Cu2+ and Mn2+ to understand superoxide radical scavenging and dismutation reactions. These complexes have been found to be efficient scavengers of O2.− (k~107–109 M−1 s−1). The effects of nature of metal ion and ligand, and stoichiometry of complex on scavenging reaction rate constants are reported. Higher scavenging rate constants have been observed with complexes of: (1) Cu2+ as compared to Mn2+, and (2) at [ligand]/[metal] ratio of one as compared to two. A clear evidence of O2.− dismutation by free metal ions and some of the complexes has been observed. The study suggests that complexes of Cu2+ and Mn2+ with small natural ligands can also act as SOD mimics.

1. Introduction Superoxide radical anion (O2.−) is produced in the physiology in cellular metabolism where oxygen scavenges electrons leaked from cellular redox processes and/or electron transport chain in addition to phagocytosis (Halliwell and Gutteridge, 2009). Further, it is a primary free radical produced in radiolysis as well as photolysis of the aerated solutions (Spinks and Woods, 1991). It is produced when dissolved oxygen scavenges radiation–generated electron in radiolysis as well as when oxygen abstracts electron from excited state of molecule in photolysis. It is a hydrophilic, rather unreactive species (E° O2,aq/ O2.−,aq =-0.18 V) which protonates to form oxidizing hydroperoxyl radical (E° HO2·, H+/H2O2 =+1.05V) in acidic medium and has a pKa at 4.8. It can be further reduced to H2O2 as suggested by the reduction potential value (E° O2.−, 2H+/H2O2 =+0.89 V) (Wood, 1988; Koppenol and Stanbury, 2010). In-vivo, O2.− is catalytically converted by superoxide dismutase (SOD) enzyme. Therefore, biological role of SOD is very important in preventing oxidative damage, inflammation, hyperoxic lung damage, atherosclerosis, and other disease pathologies induced by O2.−. The catalytic center of SOD enzymes contain a transition metal ion (Cu/ Mn/ Fe), which can undergo sequential reduction and re-oxidation to convert O2.− to H2O2 in a ping-pong mechanism (Eqs. (1) and (2)) (Klug-Roth et al., 1973; Rabani et al., 1973; Fielden et al., 1974; Klug-

E-mail address: [email protected]. http://dx.doi.org/10.1016/j.radphyschem.2017.05.010 Received 18 January 2017; Accepted 12 May 2017 Available online 18 May 2017 0969-806X/ © 2017 Elsevier Ltd. All rights reserved.

Roth and Rabani, 1976; Cabelli and Bielski, 1984a, 1984b; Barnese et al., 2012) followed by conversion of H2O2 in to H2O and O2 by catalase enzyme (Eq. (3)). M(n+1)+ + O2·− → (Mn+ + O2) or MOOn+

(1)

Mn+ / MOOn+ + O2·− + 2H+ → M(n+1)+ + H2O2 + –/ O2

(2)

H2O2 → H2O + (1/2)O2

(3) O2.−

(E° The reduction potential values for conversion of O2 into O2,aq/ O2.−aq =-0.18V) and reduction of O2.− into H2O2 (E° O2.−, 2H+/H2O2 +0.89 V) suggest that any species having redox potential in between these two values can act as SOD mimic (Koppenol and Stanbury, 2010; Sawyer and Valentine, 1981; Wood, 1988). This concept has been reported in the literature using free metal ions (Mn2+ and Cu2+) and their complexes for scavenging and dismutation of O2.− (Barnese et al., 2008; Batinic-Haberle et al., 2010; Duncan and White, 2012; Holm et al., 1996; Iranzo, 2011; Jacobsen et al., 1997; Joseph and Nagashri, 2012; Klug-Roth and Rabani, 1976; Melov et al., 2001; Miriyala et al., 2012; Rabani et al., 1973; Salvemini et al., 2002; Santini et al., 2014). Since the complexation with inorganic and organic ligands affect the reduction potentials of metal ions (Mn2+ and Cu2+), therefore, metal–ligand complexes can be tailored to tune their redox potentials in the range, +0.89 V > E° > −0.18 V, to make them

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of natural origin (natural products and biomolecules) have been suggested to function as SOD mimics (Selvaraj et al., 2014). In this context, SOD mimicking activity of Cu2+ and Mn2+ metal ions in presence and absence of some natural organic ligands at neutral pH has been investigated. The kinetics and mechanism of scavenging of O2.− radical anion by metal ions, metal ion–ligand system, and dismutation of O2.− radical anion by the in-situ produced transient species have been studied explicitly using pulse radiolysis technique. The effects of nature of metal ion, ligand, and stoichiometry of metalligand complex on scavenging and dismutation of O2.− radical have been studied and reported in this article. It is to be noted that the reaction of O2.− with metal center and/or ligand can be distinguished by such direct method only. Further, a comparison of similar scavenging process reported in the literature for some Cu and Mn complexes with other ligands has also been presented.

mimics of SOD (Barnese et al., 2012; Batinic-Haberle et al., 2010; Cabelli and Bielski, 1984;Sawyer and Valentine, 1981). In general, Mn2+ and Cu2+ based complexes have been investigated because these metal ions are naturally present as catalytic centers in SODs and are less toxic for normal cells at lower concentrations. Incidentally, pharmacological activity of some anti-inflammatory agents is also suggested to be due to their Cu complexes formed invivo. Therefore, similar metal complexes with various ligands are assumed to have potential as SOD mimics for therapeutic purpose (Duncan and White, 2012; Iranzo, 2011; Joseph and Nagashri, 2012; Melov et al., 2001; Miriyala et al., 2012; Salvemini et al., 1999, 2002; Santini et al., 2014). Using the concept of SOD mimics, complexes of metal ions with macrocyclic ligands as functional mimics of SOD have been studied earlier (Duncan and White, 2012; Durot et al., 2005; Pei et al., 2013; Riley, 1999; Salvemini et al., 1999, 2002; Santini et al., 2014). However, the same criteria that make these metal–macrocyclic complexes as potent SOD mimics, also allow them to reduce other reactive species and interfere in biological processes (Batinic-Haberle et al., 2010). Moreover, the use of SOD and catalase as therapeutic agents to decrease ROS–induced damages has shown mixed results (Duncan and White, 2012; Iranzo, 2011; Lardot et al., 1996; Melov et al., 2001; Miriyala et al., 2012; Pei et al., 2013; Salvemini et al., 1999, 2002; Santini et al., 2014; Simonson et al., 1997). On the other hand, metal ions in food are present in complexed form with natural ligands. Further, free metal ions can form complexes with various organic molecules (as ligands) in a stoichiometry depending on [ligand]/[metal] ratio, pH, etc. The metal–ligand complexes are generally formed in a fixed stoichiometry but a dynamic equilibrium between metal ion–ligand complex and free ligands can exist at all concentrations in dilute solutions. Such situations may exist physiologically where metal ions are present in presence of biomolecules and/or natural products. These complexes of metal ions with small molecules

Catechol

2. Experimental 2.1. Chemicals The ligand molecules namely catechol (1,2-dihydroxybenzene), gentisic acid (2,5-dioxybenzoic acid), tetrahydroxyquinone, tryptophan ((2S)-2-amino-3-(1H-indol-3-yl)propanoic acid), tyrosine (4-hydroxyphenylalanine), embelin (2,5-dihydroxy-3-undecyl-2,5-cyclohexadiene1,4-dione) and bilirubin of high purity were purchased from SigmaAldrich and used as received (Scheme 1). All other chemicals were of analytical reagent grade. Nanopure water (conductivity, 0.06 μS cm−1) was used for preparing solutions. Ligand and metal ion solutions have been mixed in concentration ratio of 1 and 2 to get presumably ML and ML2 stoichiometry, respectively at pH 7 in phosphate buffer solution.

Gentisic acid

Tryptophan

Tetrahydroxyquinone

Tyrosine

Embelin Bilirubin Scheme 1. Structure of ligands.

75

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Table 1 a Rate constants (108 M−1 s−1) for reaction of Cu2+ and Mn2+ complexes (MLn, n=1,2) with O2·− radical at pH 7. Ligand

Cu2+ (1:1)

Cu2+ (1:2)

Mn2+ (1:1)

Mn2+ (1:2)

Gentisic acid Catechol Tyrosine Tryptophan THQ Embelin Bilirubin

14.6 1.44 9.18 5.53 6.30 1.55$ ND

10.8 0.51 5.54 4.11 3.52 ND ND

0.92 1.52 0.70 1.00 1.50 0.27 0.98

0.56 1.13 0.65 0.62 0.84 ND ND

a Rate constants for metal ions with O2·− radical are (Bielski et al., 1985): Cu2+→ Cu+8.1×109 M−1 s−1 (pH7); Cu+→ Cu2+1×1010 M−1 s−1 (pH3-6.5). Mn2+→ Mn3+0.13×108 M−1 s−1 (pH7.3); Mn2+→ MnOO+1.1×108 M−1 s−1 (pH6.7). $ k(Cu2Emb + O2·−)=3.27×109 M−1 s−1, ND → not detected.

2.2. Pulse radiolysis Fig. 1. Kinetic absorption traces at 270 nm after repetitive electron pulses to aerated aqueous solution containing phosphate buffer (2 mM), HCOONa (0.04 M). Solution was shaken in air after 10th and 19th pulses only, dose 107 Gy/pulse.

Linear electron accelerator giving pulses of 7 MeV electrons was used as a radiation source for in-situ generation of superoxide radical. High-energy electrons deposit energy in water to generate its free radicals and molecular species (Eq. (4)). H2O ~~~~→ e¯aq (0·27), H3O+ (0·27), H·(0·062),·OH (0·28), H2 (0·047), H2O2 (0·073)

concentration ratios to get presumably ML and ML2 complexes. Complexes of other stoichiometry could be present in dynamical equilibrium but experimental results do not indicate the presence of either free metal ion or free ligands. Therefore, it is safe to assume the presence of ML and ML2 complexes under the experimental conditions. The prepared solutions were clear, colorless and without precipitation. Catechol (Cat), gentisic acid (GA), tetrahydroxyquinone (THQ), tryptophan (TrpH), tyrosine (TyrOH), embelin (Emb) and bilirubin (BR, a tetrapyrrole with two –COOH groups) have been used as natural ligands for complexation with Cu2+ and Mn2+ ions. These molecules have been selected as they represent phenol, quinone, alkyl-quinone, amino acid, and complex tetrapyrrole ligands. Transient absorption spectra and scavenging rate constant of O2.− radical by the metal–ligand complexes ([MLn]) were measured with aerated (0.25 mM) aqueous solution and radiation dose up to 20 Gy/ pulse (12 μM [CO2.−]). The rate constants measured for the reactions of O2.− scavenging by Cu2+ and Mn2+ complexes with above mentioned ligands in the present study are given in Table 1. The calculation of rates using formation traces and second order rate constant has been shown using example of THQ ligand with Mn2+ metal ion in Figs. S1. It has been noticed that repetitive electron (radiation) pulses to aerated aqueous solution (0.25 mM) without metal–ligand complex results in loss of dissolved oxygen (Fig. 1). By repetitive fifth electron pulse (cumulative [CO2.−] radical ~ 0.3 mM) there is insufficient oxygen remained in aerated aqueous solution for formation of O2.− radical and a clear decay of CO2.− radical has been observed on tenth electron pulse (Fig. 1). Further, shaking of the oxygen-depleted solution in the air (after 10th and 19th pulse) produced almost same concentration of O2.− radical on next pulse (11th and 20th) of electron beam radiation (Fig. 1). Therefore, oxygen saturated solutions (1.4 mM [O2]) have been used for experiments using repetitive electron pulses with dose up to 100 Gy/ pulse (60 μM [CO2.−]). In general, (1) formation of transient/s has been studied at low radiation dose and high solute concentration, and (2) dismutation of O2.− radical by in-situ formed metal-ligand transient has been studied at high radiation dose and [MLn] similar to or less than [O2.−].

(4)

The values in parenthesis are radiation chemical yields (μmol/ Joule) of the species. An air-saturated solution containing 5.0×10−2 mol dm−3 KSCN (Gε =2.6×10−4 m2 J−1 at 475 nm) was used to measure absorbed radiation dose (Buxton and Stuart, 1995). The optical path length was 1.0 cm and detection system covered the wavelength range ~250–750 nm. The calculated rate constants and wavelength maximum contain experimental error of 10% and 5 nm, respectively. Aerated (0.25 mM O2) or oxygen (> 99% purity)- saturated (1.4 mM O2) aqueous solution containing excess formate anion (0.04 M) has been used to convert all reactive primary radicals of water in to O2.− exclusively (Eqs. (5)–(7)). ·OH/H· + HCO2− → CO2·− + H2O/H2

CO2·− + O2 → CO2 + O2·−

(k=3·5/0·13×109 M−1 s−1) (5)

(k=2·4×109 M−1 s−1)

− e¯ (k=2·0×1010 M−1 s−1) aq + O2 → O2·

(6) (7)

Aqueous solutions of Na2HPO4 and NaH2PO4 in equal concentrations (2 mM) have been used to make pH 7 solution. A small change in pH by addition of other solutes was adjusted with dilute HClO4/NaOH solution. The transient spectra and rate constants measurement have been done using 100 ns electron pulse with dose up to 20 Gy/ pulse. Formation rates of solute transient for Mn2+(at 300 nm) or decay rates of reacting radical (O2.−, 270 nm) for Cu2+ have been measured at 3–4 solute concentrations. These rates (k, s−1) have been plotted against solute concentration (M) to get second order scavenging rate constant (M−1 s−1). A representative example for measurement of O2.− scavenging rate and calculation of rate constant is shown in Fig. S1. Longer electron pulse width of 500 ns and 2 μs have been used to deliver larger radiation doses in the range of ~30 Gy and ~110 Gy, respectively. The oxygen present in aerated (0.25 mM O2) and oxygen-saturated (1.4 mM O2) aqueous solution is sufficient to scavenge CO2.− radical produced at 20 Gy (12 μM) and 100 Gy (60 μM), respectively.

3.1. Scavenging and dismutation of O2.− radical 3.1.1. Cu2+-GA (1:1) In addition to metal-ligand complex, free ligand and free metal ion may also react directly with O2.− radical. Therefore, scavenging reactions of O2.− radical with free ligand, free metal ions, and complexes of metal ions (Cu2+ and Mn2+) with one of the ligand (gentisic acid, GA) have been studied in detail. Gentisic acid has been

3. Results and discussion As mentioned earlier, metal (M) ion and ligands (L) have been taken in 2 mM phosphate buffered aqueous solution (pH 7) in 1:1 and 1:2 76

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(Schroeder et al., 1966). This negative absorption at higher time points, however, almost disappears by 8th electron pulse. The decay traces in inset of Fig. S2 show that Cu+(280 nm) formed in the initial pulses is re-oxidized by O2.− radical produced in the later pulses causing dismutation of O2.− radical. The measured decay rates of Cu+ increase from ~4.2×104 s−1 at 2nd pulse to ~8.0×104 s−1 8th pulse, probably due to accumulation of Cu+ ion with radiation pulses. The latter process (re-oxidation of Cu+) could be taking place since no formation of either copper nanoparticle or copper oxide has been observed (visually). However, reaction of Cu2+-GA (1:1) (0.1 mM) with O2.− (19.8 μM) produced transient absorption spectrum having maxima at 250 nm, a shoulder at 280 nm and an absorption band at 320 nm (Fig. 3A). A comparison with transition absorption bands mentioned above and decay traces of transient absorptions (inset of Fig. 3A) suggests that absorption at 240–280 nm is due to Cu+-GA formed by electron transfer from O2.− radical (k=1.46×109 M−1 s−1) whereas 320 nm transient absorption band may be due to addition of O2.− with GA of Cu2+-GA complex (i.e., Cu2+-GA-O2.−). Further, the transient absorption at 320 nm in Fig. 3A is not due to GA-electron adduct (GA.− radical) which has absorption bands at 360 and 440 nm (Fig. 2B). The decay profiles of Cu+-GA (absorption at 280 nm) and Cu2+-GA-O2.− radical adduct (absorption at 320 nm) have been studied by monitoring absorption at 280 and 320 nm as shown in Fig. 3B, C but at higher doses of 95 Gy/ pulse. The decay of Cu+-GA complex (absorption at 280 nm) formed in the reaction of Cu2+-GA complex (400 μM) with O2.− radical has been found to be same from 1st (corresponding to 57 μM) to 10th (corresponding to 570 μM) electron pulse (Fig. 3B). This suggests that in-situ formed Cu+-GA complex scavenges O2.− radical to regenerate Cu2+-GA complex resulting in no loss of complex concentration. Further, similar absorption decay pattern even with multiple electron pulses at 320 nm (Fig. 3C) suggests insignificant loss of complex concentration. The second step of reaction of O2.− radical with in-situ formed Cu+GA transient complex is also evident on comparing transient absorption profiles obtained on giving repetitive electron pulses to a solution of GA (inset of Fig. 2A), Cu2+(inset of Fig. S2) and Cu2+-GA (1:1) (Fig. 3B and C). It has been assumed that Cu+-GA complex formed with successive electron pulses accumulates due to sufficient stability in water (Eq. (8)) that is followed by its reaction with O2.− radical produced in later electron pulses (Eq. (9)). The decay traces in Fig. 3B shows that Cu+-GA transient formed in the initial pulses reacts with O2.− radical produced in the later pulses causing dismutation of O2.− radical with k~4.6×104 s−1 (1st T1/2~15 μs). However, reaction of ligand GA (0.2 mM) alone with cumulative concentration of O2.− radical (57 μM/pulse) show some loss of GA (Fig. 2A inset). GA and Cu2+ react with O2.− radical with bimolecular rate constants of < 104 M−1 s−1 (present work) and 8.1×109 M−1 s−1 (Bielski et al., 1985), respectively. However, the Cu2+-GA complex has been found to react with O2.− radical with a second order rate constant of 1.46×109 M−1 s−1. As mentioned earlier, kinetic absorption traces observed in the reaction of O2.− radical with Cu2+(Fig. S2 inset), GA (Fig. 2A inset) and Cu2+-GA (1:1) complex (Fig. 3B, C) suggest that the copper complex catalyze the dismutation O2.− radical in two steps like SOD enzyme (Eqs. (8) and (9)).

Fig. 2. (A) Transient absorption spectrum obtained from O2-saturated aqueous solution containing gentisic acid (0.2 mM), phosphate buffer (2 mM) and HCOONa (40 mM) at 20 μs, 50 μs, and 80 μs after electron pulse (100 Gy/ pulse). Inset: Kinetic absorption traces at 320 nm after nth number of repetitive electron pulses (95 Gy/ pulse) to gentisic acid (50 μM) under similar conditions. (B) Transient absorption spectrum obtained from N2-bubbled (de-aerated) aqueous solution containing gentisic acid (0.2 mM), phosphate buffer (4 mM) and tert-butanol (0.2 M) at 10 μs and 300 μs after electron pulse (15 Gy/ pulse).

used as a representative example for discussing reactions of ligand–metal ions (Cu2+ and Mn2+) complexes used in this study. Reaction of GA (0.2 mM) with O2.− radical (60 μM) produced a strong transient absorption below 300 nm with only a shoulder at 320 nm (Fig. 2A), which could be due to solute-radical adduct (GAOO.−) formation. This has been assumed because reaction of GA (0.2 mM) alone with hydrated electron (4.2 μM [eaq−]) produced transient absorption bands at 360 nm and 440 nm (Fig. 2B), which could be due to formation of solute radical anion (GA.−). The reaction of Cu2+(0.1 mM) alone with O2.− radical (19.8 μM) produced transient absorption spectrum having a maximum at 250 nm with a shoulder at 280 nm (Fig. S2). The absorption band at 280 nm for Cu+ observed in the reaction of Cu2+ alone with O2.− radical has been used to study catalytic dismutation of O2.− radical by Cu2+ alone. Cu2+(0.2 mM) reacted with O2.− radical to produce almost same kinetic absorption traces from 2nd pulse (~0.12 mM O2.− radical) to 8th pulse (~0.48 mM O2.− radical) at 280 nm except a small difference in absorption at higher time points (Fig. S2 inset). The absorption profile at 280 nm has been found to be a little below the baseline at higher time points after 2nd pulse (inset of Fig. S2) which is due to formation and presence of Cu+ ion in solution. It is to be noted that absorption bands of Cu+ and Cu2+ are reported to have overlapping absorption bands with maxima at ~275 nm (ε=1300 M−1 cm−1) and 280 nm (ε=2900 M−1 cm−1), respectively (Zafiriou et al., 1998). Further, Cu+ complexes are reported to be sufficiently stable in water

Cu2+-Ln + O2·− (270 nm) → Cu+-Ln (280 nm) + O2 (k=1·46×109 M−1 s−1)

(8)

Cu+-Ln (280 nm) + O2·− (270 nm) → Cu2+-Ln + O22¯ + O2 (k=4·6×104 s−1, T1/2~15 μs)

(9)

3.1.2. Cu2+/Mn2+-BSA (1:1) Metal ions also bind to macromolecules, similar to that with small organic ligands, forming complexes. In physiology, reaction of these 77

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Fig. 3. (A) Transient absorption spectrum obtained on pulse radiolysis (33 Gy/ pulse) of air saturated aqueous solution containing Cu2+-gentisic acid (0.1 mM each), phosphate buffer (2 mM), HCOONa (50 mM) after 2 μs, 10 μs and 20 μs. Inset: Kinetic absorption traces at 240 nm, 280 nm and 320 nm under identical conditions. Kinetic absorption traces after multiple electron pulses from O2-satuated aqueous solution containing phosphate buffer (2 mM), HCOONa (50 mM) and (B) Cu2+-gentisic acid (0.4 mM each) at 280 nm, 100 Gy/ pulse and (C) Cu2+-gentisic acid (0.05 mM each) at 320 nm and 95 Gy/ pulse.

metal ion-macromolecule complexes with O2.− radical is also inevitable. In an earlier study, complex of Cu2+ with bovine serum albumin (BSA) protein, a macromolecule was reported to dismutase O2.− radical with rate constant of 3.7×106 M−1 s−1 (Plonka et al., 1991). It is reported that Cu2+-BSA reacts with O2.− radical to produce transient absorption band with maximum at 420 nm (disulfide radical anion of BSA) and absorption below 300 nm (Cu+) (Plonka et al., 1991). In the present study, kinetic absorption profiles for reaction of 30 μM Cu2+-BSA (1:1) with cumulative O2.− radical concentration from 43 μM (1st pulse) to 430 μM (10th pulse) have been found to be almost same (figure not shown). This suggests no loss of (Cu2+-BSA) complex due to catalytic dismutation (~2.5×103 s−1, 1st T1/2~ 270 μs) of O2.− radical through formation of Cu+-BSA. Similarly, It has been found that Mn2+-BSA (1:1) complex scavenges O2.− radical to produce transient absorption bands at ~420 nm and ~300 nm (Fig. 4A). Kinetic absorption traces at these two wavelengths shown in the inset of Fig. 4A suggest a fast decay of BSA radical anion (420 nm) and a slower formation of absorption at 300 nm. The transient absorption band at 300 nm has been assigned to MnOO+-BSA transient and the reasons are discussed below. However, a clear transformation from absorption band at 420 nm (BSA.−) to 300 nm (MnOO+-BSA transient) has not been observed. Therefore, it can be assumed that MnOO+-BSA transient (300 nm absorption band) has been formed by direct reaction of O2.− radical with Mn2+-BSA, which is slower than decay of BSA.−. Further, kinetic absorption traces at 300 nm in the reaction of Mn2+-BSA (30 μM) with cumulative O2.− radical concentration from ~56 μM (1st pulse) to ~560 μM (10th pulse) are almost

same suggesting catalytic dismutation of O2.− radical through intermediacy of MnOO+-BSA with no loss of reactant Mn2+-BSA (Fig. 4B). The bimolecular rate constant for formation of absorption band at 300 nm with Mn2+-BSA has been found to be ~3.5×107 M−1 s−1, which is an order of magnitude more than that with Cu2+-BSA (3.7×106 M−1 s−1) reported by Plonka et al. The rate constant for scavenging of O2.− radical by MnOO+-BSA has been found to be ~3.2×103 s−1 (1st T1/2~ 215 μs). The experimental observation of almost same absorption traces on multiple electron pulses producing large concentration of O2.− radical (43 μM to ~560 μM) to small concentration of Cu2+/Mn2+-BSA (30 μM) complex shows scavenging of O2.− radical by Cu2+/Mn2+BSA complex and dismutation of O2.− radical by Cu+/MnOO+-BSA transient. 3.1.3. Mn2+-GA (1:1) Mn2+ and its inorganic complexes are known to react with O2.− radical to produce absorption band in 260–275 nm region, which has been attributed to MnOO+ transient (Cabelli and Bielski, 1984; Barnese et al., 2012; Bielski et al., 1985). In the present work, reaction of Mn2+(~M) with O2.− radical (~10 μM) in both (1) 1 M aerated ethanol, and (2) aerated aqueous solution of formate anion at pH 7 produced transient absorption bands at 270–275 nm due to formation of MnOO+ transient (Fig. S3, S4A). The strong absorption band at 270–275 nm (MnOO+) allows us to study catalytic dismutation of O2.− radical by Mn2+ complex. In the reaction of Mn2+ alone (200 μM) with O2.− radical almost same kinetic 78

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Fig. 4. (A) Transient absorption spectrum obtained on pulse radiolysis (17 Gy/ pulse) of air saturated aqueous solution containing Mn2+-BSA (86 μM each), phosphate buffer (2 mM), HCOONa (50 mM) after 200 μs and 600 μs. Inset: Kinetic absorption traces at 300 nm and 420 nm under identical conditions. (B) Kinetic absorption traces after multiple electron pulses from O2-satuated aqueous solution of Mn2+-BSA (30 μM each) at 300 nm under identical conditions and at 94 Gy/ pulse.

Fig. 5. (A) Transient absorption spectrum obtained on pulse radiolysis (16 Gy/ pulse) of air saturated aqueous solution containing Mn2+-gentisic acid (0.4 mM each), phosphate buffer (2 mM), HCOONa (40 mM) after 4 μs, 34 μs and 82 μs. Inset: Kinetic absorption traces at 270 nm for [Mn2+-gentisic acid (1:1)] (μM) under identical conditions. (B) Kinetic absorption traces for the same solution at 280 nm, 300 nm, 350 nm and 410 nm under identical conditions.

absorption traces have been observed from 1st pulse (~57 μM O2.− radical) to 20th pulse (~1.14 mM O2.− radical) in aqueous-ethanol solution (Fig. S3 inset), and from 1st pulse (~64 μM O2.− radical) to 20th/22nd pulse (~1.28 mM/~1.41 mM O2.− radical) in aqueous solution (Fig. S4B, S4C). The reaction of Mn2+ with large concentration of O2.− radical on repetitive electron pulses shows that MnOO+ formed in the initial electron pulses reacts with O2.− radical produced in the later pulses causing its dismutation with k~1.5×104 s−1 (1st T1/ 2~46 μs). In this case also, reaction of Mn2+-GA (1:1) complex with O2.− radical has been studied in detail as a representative example of Mn2+ligand complexes. The reaction of Mn2+-GA (1:1) complex (400 μM) with O2.− radical (9.6 μM) has produced transient absorption bands at 260–310, 410–430 nm (Fig. 5A), which could be due to MnOO+-GA transient. The kinetic absorption traces for the formation of MnOO+GA transient at 270 nm are shown as inset of Fig. 5A with increasing concentrations (80 μM, 200 μM and 400 μM) of Mn2+-GA (1:1) complex to show the complex concentration effect. The observed absorption bands for Mn2+-GA with O2.− radical are blue shifted as compared to the absorption bands of GA-electron adduct (GA.−), which has been observed at 360 and 440 nm (Fig. 2B). Absorption band of MnOO+-GA has been also observed at a different position as compared to that reported earlier for MnOO+(260–275 nm) (Barnese et al., 2012; Bielski et al., 1985; Cabelli and Bielski, 1984) and observed in the present work (Figs. S3, S4). Further, transient absorption spectrum observed in the reaction of GA with O2.− radical (310 nm, Fig. 2A) is also different as compared to that of Fig. 5A. Therefore, absorption bands at ~310 nm and ~430 nm can be ascribed to MnOO+-GA transient with absorption

contribution of (1) O2.− radical below 350 nm at initial time points and (2) O2.− radical adduct with GA of Mn2+-GA complex (Mn2+-GA-O2.−) beyond 350 nm. The absorption of MnOO+-GA transient has been used to study its kinetics to get simultaneous decay of O2.− radical and formation of MnOO+-GA transient. A plot of kinetic absorption traces at different wavelengths clearly suggests simultaneous decay of O2.− radical (up to ~3 μs) at lower wavelengths and formation of MnOO+-GA transient (after 3 μs) at higher wavelengths (Fig. 5B). The initial decay of absorption could be seen only up to ~360 nm which could be attributed to absorption of high concentration of O2.− radical (~10 μM) which decay by ~3 μs on reaction with Mn2+-GA (1:1) complex to produce MnOO+-GA transient (absorption 250–450 nm). Further, reaction of MnOO+-GA transient with O2.− radical has been studied using repetitive electron pulses given to Mn2+-GA (1:1) complex (400 μM) with higher radiation dose and O2-saturated (1.4 mM) condition producing ~52 μM [O2.−]/ pulse (Fig. 6). In O2-saturated solution, kinetic absorption traces for formation as well as decay at 310 nm (and 430 nm) are almost same even after repetitive pulses. The concentration of O2.− radical (~52 μM/pulse) and k(Mn2+-GA + O2.−) value (0.92×108 M−1 s−1) suggest that completion of ~90% of reaction (2nd order) of Mn2+-GA with O2.− radical requires ~1850 μs time (9/k[C]). Therefore, growth in absorption at shorter time up to 100 μs (for 310 and 430 nm) and decay in absorption in longer time domain up to 5 ms (for 310 and 430 nm) are due to formation of MnOO+-GA and reaction of O2.− radical with MnOO+-GA, respectively (Fig. 6). However, decay 79

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Fig. 6. Kinetic absorption formation and decay traces obtained on pulse radiolysis (87 Gy/ pulse) of O2-saturated aqueous solution containing Mn2+-gentisic acid (0.4 mM each), phosphate buffer (2 mM), HCOONa (40 mM) at 310 nm and 430 nm on multiple electron pulses.

trace at 310 nm after tenth pulse shows formation of some product formation (Fig. 6). The absorption decay at 430 nm allows us to measure value of rate (k) for MnOO+-GA reaction with O2.− radical (3.6×103 s−1, 1st T1/2~200 μs). These observations suggest that Mn2+-GA (1:1) complex reacts with O2.− radical in two steps to bring about its dismutation, like SOD enzyme (Eqs. (10) and (11)). Mn2+-Ln + O2·− → MnOO+-Ln

(0·92×108 M−1 s−1)

MnOO+-Ln + O2·− → Mn2+-Ln + O22¯ + O2 (3·6×103 s−1, 1st T1/2~200 μs)

(12b)

Mn2+-Ln + O2·− → MnOO+-Ln

(13a)

MnOO+-Ln + O2·− → Mn2+-Ln + O22¯ + O2

(13b)

This ping-pong mechanism of reduction of metal centers followed by re-oxidation of transient/ intermediate has been shown earlier for SOD enzymes and other such complexes (Klug-Roth et al., 1973; Rabani et al., 1973; Fielden et al., 1974; Klug-Roth and Rabani, 1976; Cabelli and Bielski, 1984a, 1984b; Barnese et al., 2012). The first step of reaction of metal ion with O2.− radical agrees well with change in electronic configuration in going from initial oxidation state to final state. On accepting one electron, Cu2+ goes (4d9 state) to more stable Cu+ state (4d10, favorable process) whereas Mn2+(4d5 state) goes to less stable Mn+ state (4d6, unfavorable process). Further, involvement of Mn3+ has not been considered in Eq. (13a) since O2.− radical having lower reduction potential (E° O2,aq/ O2.−,aq =-0.18V) than Mn3+(E° Mn3+/Mn2+=+1.5V) cannot oxidize Mn2+ to Mn3+. Similarly, involvement of Cu+ is considered (Eq. (12)) because reduction potential value of Cu2+/Cu+(E° Cu2+/Cu+=+0.159) is greater than that for O2.− radical. Further, involvement of Cu+ is assumed as it can revert to more stable Cu2+ in aqueous solution probably for gain in hydration energy. It is to be noted that the reported hydration energies of Cu2+ and Cu+ are −2161 and −610 kJ/mol, respectively (RSC Data Book, 2017).

(10)

(11)

3.2. Mechanistic aspects The kinetic absorption traces recorded for the reaction of O2.− radical with metal ions (Cu2+, Mn2+) as well as metal-ligand complexes on repetitive electron pulses are almost same from 1st pulse (≥20 μM O2.− radical) to nth pulse (cumulative ≥20 times ‘n′ μM O2.− radical) in O2-saturated system in the present study (Insets of Fig. S2 and S3, Figures S4B, S4C, 3B, 4B, 6). This suggests that metal ions are not consumed in this process under the present experimental conditions probably due to catalytic dismutation reaction. It is further evident from the kinetic absorption traces that transient absorption method could not differentiate between reactions of O2.− radical with Cu2+ and Cu+ ions (inset of Fig. S2). It can be said that dismutation reaction of O2.− radical with Cu2+ and Mn2+ takes place via formation of Cu+ and MnOO+ transients, respectively in presence of both with inorganic (anions) and organic ligands (Eqs. (12) and (13)). Cu2+-Ln + O2·− → Cu+-Ln + O2

Cu+-Ln + O2·− → Cu2+-Ln + O22¯ + O2

3.3. A comparison of rate constants A comparison of reported rate constants shows that scavenging rates of O2.− radical by copper (8.1×109 M−1 s−1 for Cu2+→ Cu+ and 1.0×1010 M−1 s−1 for Cu+→ Cu2+) are higher than those values

(12a) 80

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reported for manganese (1.1×108 M−1 s−1 for Mn2+→ MnO2+ and 1.3×107 M−1 s−1 for Mn2+→ Mn3+) (Bielski et al., 1985). Further, the reported rate constants for scavenging of hydrated electron (eaq-) by copper (Cu2+: 3.3×1010 M−1 s−1 and Cu+: 2.7×1010 M−1 s−1) are also greater than those reported with manganese (Mn2+: 2.0×107 M−1 s−1 and Mn3+-ligands: ~ 1010 M−1 s−1) (Buxton et al., 1988). The present study shows that O2.− radical scavenging rates are higher with free metal ion as well as complex of Cu2+ as compared to those with Mn2+(Table 1 and footnote). It has been noticed on comparing the measured rate constants (present study) for the reaction of O2.− radical with ML and ML2 complexes that, in general, ML complex is more efficient scavenger of O2.− radical than ML2 for Cu2+ as well as Mn2+ ion (Table 1). Further, the rate constants for Cu2+ complexes ([L]/[M]=1 and 2) with studied ligands are in the order: GA > TyrOH > others. Similarly, the rate constants for Mn2+ complexes ([L]/[M]=1 and 2) are in the order: Cat > THQ > others. The O2.− radical scavenging rate constants by ML and ML2 are 3–4 orders of magnitude greater than those reported for the corresponding ligand only (Bielski et al., 1985). Further, O2.− radical scavenging rate constants for the studied Cu2+ complexes (~ 108–109 M−1 s−1) are much less than that for free Cu2+ ion (8.1×109 M−1 s−1) (Bielski et al., 1985). However, O2.− radical scavenging rate constants by the studied Mn2+ complexes (~107–108 M−1 s−1) are less than or of the same order as Mn2+ ion (1.1×108 M−1 s−1) only (Bielski et al., 1985). The order of O2.− radical scavenging rate constants by metal ions, ligands and complexes can be summarized as in Eqs. (14) and (15): k (Cu2+ + O2·−) > k (Cu2+-L + O2·−) > k (Cu2+-L2 + O2·−) > > k (L + O2·−)

dismutation rate of O2.− radical is slowest with BSA ligand but is almost same with free ion and with GA ligand. It can be inferred from these observations that free metals are highly active for dismutation reaction and complexation decreases this activity. Furthermore, kinetics absorption traces for dismutation reaction of O2.− radical with Mn2+-BSA/ GA/- and Cu2+-BSA/GA/- suggest that this is a metal-centric process producing MnOO+-Ligand and Cu+-Ligand, respectively. Therefore, it can be inferred that Mn2+ and Cu2+ complexes with other ligands also dismutase O2.− radical after the first step of scavenging it. It is to be noted that the Cu2+ complexes ([L]/[M]=1,2) with highest scavenging rate constant for O2.− radical have been found with those ligands, which have –OH and –COOH groups on the same molecule (gentisic acid and tyrosine). However, Mn2+ complexes with highest scavenging rate constant for O2.− radical have been found with those ligands, which have two ortho –OH groups on the same aromatic ring (catechol and tetrahydroxyquinone). This selectivity of ligands suggests role of specificity of metal-ligand complex to allow reaction of metal ion center with O2.− radical. In this context two more ligands (embelin and bilirubin) have been used to make Mn2+ and Cu2+ complexes. Embelin having two quinonic and two hydroquinone substituents has been selected as a specific bidentate ligand, which has an additional hydrophobic group. The measured O2.− radical scavenging rate constants (k, 109 M−1 s−1) for reaction of O2.− radical with Cu2+ complex have been found to vary as: 8.10 (Cu2+) > 3.27 (2Cu2+-Emb) > 0.15 (Cu2+-Emb) suggesting loss of interaction site with increasing number of ligands. The measured O2.− radical scavenging rate constants for Cu2+/Mn2+-embelin (1:1) are among the smallest and could not be detected for Cu2+/Mn2+embelin2 (1:2), which could be due to rigid structure of ML2 (1:2) complex and hydrophobic nature of complex. Another selected ligand, bilirubin, has four tetrapyrrole groups and two carboxylic groups to study the effect of multidentate ligand on O2.− radical scavenging reaction (Table 1). Interestingly, no reaction of O2.− radical with Cu2+ complex has been observed with bilirubin ligand, which has six coordinating atoms for ‘soft’ Cu2+ ion including four ‘soft’ donor atoms (nitrogen) of pyrrole groups in addition to two –COOH groups. Similarly, no reaction of O2.− radical with Cu2+/Mn2+-embelin2/ bilirubin2 has been detected. These observations suggest that the electron transfer from O2.− radical to complex needs specific geometry of complex to allow approach of O2.− radical to the metal ion and does not occur through outer-electron transfer process.

(14)

k (Mn2+-L + O2·−) ≥ k (Mn2+ + O2·−) > k (Mn2+-L2 + O2·−) > > k (L + O2·−) (15) In other words, complexation of Cu2+ and Mn2+ ions modifies their O2.− radical scavenging activity, decreases for Cu2+ ions but increases for Mn2+ ions. Reaction of O2.− radical with Cu2+ and Mn2+ in absence of complexing ligands produce transients with absorption maxima at 280 nm and ~275 nm, respectively. The absorption maxima of the transients produced in the reaction of the organic ligands with O2.− and eaq− are reported in the literature (Bielski et al., 1985; Buxton et al., 1988). Therefore, a comparison of transient absorption bands observed in the reaction of Cu2+/ Mn2+ complexes, free Cu2+/ Mn2+ ions and free ligands with O2.− radical suggests the involvement of metal-ligand complex as a single unit in the studied superoxide dismutation reaction. The two steps of O2.− dismutation have not been observed clearly with Cu2+/Mn2+ complexes due to formation of single transient species, Cu+/MnOO+-ligand complexes (Eqs. (12) and (13)). As mentioned earlier that repetitive electron pulses produce almost same absorption traces for transients (Cu+-Ln or MnOO+- Ln), which suggests dismutation of O2.− radical on its reaction with transients of metal ions in presence or absence of organic ligands (Cu+-Ln or MnOO+- Ln) and regenerating reactant (Cu2+-Ln or Mn2+-Ln). The kinetic absorption traces observed at 280 nm for Cu2+ alone (inset of Fig. S2), at 280 nm for Cu2+-GA complex (1:1, Fig. 3B), at 300 nm for Cu2+-BSA complex (1:1) (figure not shown), at 270 nm for Mn2+ alone (Fig. S4B, S4C), at 430 nm for Mn2+-GA complex (1:1, Fig. 6), and at 300 nm for Mn2+-BSA complex (1:1, Fig. 4B) have been used to calculate 1st half-life of O2.− radical in dismutation process which are ~20 μs, ~12 μs, ~315 μs, ~46 μs, ~200 μs and ~215 μs, respectively. In other words, order of dismutation half-lives with Mn2+-Ln is: MnOO+-BSA complex (1:1) > MnOO+-GA complex (1:1) > MnOO+ alone; and with Cu2+-Ln is: Cu+-BSA complex (1:1) > Cu+ alone > Cu+-GA complex (1:1). In case of Mn2+, half-life of superoxide radical dismutation is shortest for pure metal ion, decrease on complexation with ligands and longest for complex with BSA protein. For Cu2+,

4. Conclusions Complexes of Cu2+ and Mn2+ with low molecular weight organic molecules of natural origin and bovine serum albumin protein have been found to scavenge and dismutate superoxide radical efficiently. The measured O2.− radical scavenging rate constants have been found to be greater with (1) Cu2+ as compared to Mn2+, and (2) at ligand to metal concentration ratio of one as compared to two. Further, O2.− radical scavenging rate constants of Cu2+ and Mn2+ complexes have been found to be greater than those with organic ligands but lesser than those with metal ions. The rate of scavenging of O2.− radical has been found to decrease with increase in size and coordination capacity of the ligand molecule. Further, the transients of metal ions (Cu+ and MnOO+) and complexes (Cu+-Ln and MnOO+-Ln) produced on scavenging superoxide radical further react with another superoxide radical in the dismutation step. The dismutation rates of O2.− radical have been found to be more with Cu2+ as compared to Mn2+ for small organic molecules as ligands. The present study also suggests that multidentate ligands like bilirubin and embelin hinder the reaction to take place between metal ion and superoxide radical. Acknowledgements Author thanks LINAC team (Mr. S.A. Nadkarni, Mrs. M. Toley and 81

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Joseph, J., Nagashri, K., 2012. Novel copper-based therapeutic agent for antiinflammatory: synthesis, characterization, and biochemical activities of copper(II) complexes of hydroxyflavone schiff Bases. App. Biochem. Biotechnology 167, 1446–1458. Klug-Roth, D., Fridovich, I., Rabani, J., 1973. Pulse radiolytic investigations of superoxide catalyzed disproportionation. Mechanism for bovine superoxide dismutase. J. Am. Chem. Soc. 95, 2786–2790. Klug-Roth, D., Rabani, J., 1976. Pulse radiolytic studies on reactions of aqueous superoxide radicals with copper(II) complexes. J. Phys. Chem. 80, 588–591. Koppenol, W.H., Stanbury, D.M., Bounds, P.L., 2010. Electrode potentials of partially reduced oxygen species, from dioxygen to water. Free Radic. Biol. Med. 49, 317–322. Lardot, C., Broeckaert, F., Lison, D., Buchet, J.P., Lauwerys, R., 1996. Exogenous catalase may potentiate oxidant-mediated lung injury in the female Sprague-Dawley rat. J. Toxicol. Environ. Health 47, 509–522. Melov, S., Doctrow, S.R., Schneider, J.A., Haberson, J., Patel, M., Coskun, P.E., Huffman, K., Wallace, D.C., Malfroy, B., 2001. Lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase–catalase mimetics. J. Neurosci. 21, 8348–8353. Miriyala, S., Spasojevic, I., Tovmasyan, A., Salvemini, D., Vujaskovic, Z., Clair St., D., Batinic-Haberle, I., 2012. Manganese superoxide dismutase, MnSOD and its mimics. Biochim. Biophys. Acta 1822, 794–814. Pei, F., He, Y., Li, K., Wang, R., Li, G., Zhao, T., 2013. SOD mimics based on macromolecules. Progress. Chem. 25, 340–349. Plonka, A., Krasiukianis, R., Mayer, J., Zgirski, A., Hilewicz-Grabska, M., 1991. Pulseradiolysis studies on reactivity of BSA and BSA-Cu(II) complexes. Radiat. Phys. Chem. 38, 445–447. Rabani, J., Klug-Roth, D., Lilie, J., 1973. Pulse radiolytic investigations of the catalyzed disproportionation of peroxy radicals. Aqueous cupric ions. J. Phys. Chem. 77, 1169–1175. Riley, D.P., 1999. Functional mimics of superoxide dismutase enzymes as therapeutic agents. Chem. Rev. 99, 2573–2587. Salvemini, D., Wang, Z.-Q., Zweiser, J.L., Samouilov, A., Macarthur, H., Misko, T.P., Currie, M.G., Cuzzocrea, S., Sikorski, J.A., Riley, D.P., 1999. A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats. Science 286, 304–306. Salvemini, D., Riley, D.P., Cuzzocrea, S., 2002. SOD mimetics are coming of age. Nat. Rev. Drug Discov. 1, 367–374. Santini, C., Pellei, M., Gandin, V., Porchia, M., Tisato, F., Marzano, C., 2014. Advances in copper complexes as anticancer agents. Chem. Rev. 114, 815–862. Sawyer, D.T., Valentine, J.S., 1981. How Super Is Superoxide? Acc. Chem. Res. 14, 393–400. Schroeder, H.A., Nason, A.P., Tipton, I.H., Balassa, J.J., 1966. Essential trace metals in man: zinc. Relation to environmental cadmium. J. Chronic Dis. 19, 1007–1034. Selvaraj, S., Krishnaswamy, S., Devashya, V., Sethuraman, S., Krishnan, U.M., 2014. Flavonoid-metal ion complexes: a novel class of therapeutic agents. Med. Res. Rev. 34, 677–702. Simonson, S.G., Welty-Wolf, K.E., Huang, Y.C.T., Taylor, D.E., Kantrow, S.P., Carraway, M.S., Crapo, J.D., Piantadosi, C.A., 1997. Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry. J. Appl. Physiol. 83, 550–558. Spinks, J.W.T., Woods, R.J., 1991. Introduction to Radiation Chemistry. John Wiley & Sons, Inc, New York. Wood, P.M., 1988. The potential diagram for oxygen at pH 7. Biochem. J. 253, 287–289. Zafiriou, C., Voelker, B.M., Sedlak, D.L., 1998. Chemistry of the superoxide radical (O2-) in seawater: reactions with inorganic copper complexes. J. Phys. Chem. A 102, 5693–5700.

Mr. S.J. Shinde) for the technical assistance during the course of the work. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.radphyschem.2017.05. 010. References Barnese, K., Gralla, E.B., Cabelli, D.E., Valentine, J.S., 2008. Manganous phosphate acts as a superoxide dismutase. J. Am. Chem. Soc. 130, 4604–4606. Barnese, K., Gralla, E.B., Valentine, J.S., Cabelli, D.E., 2012. Biologically relevant mechanism for catalytic superoxide removal by simple manganese compounds. PNAS 109, 6892–6897. Batinic-Haberle, I., Reboucas, J.S., Spasojevic, I., 2010. Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential. Antioxid. Redox Signal 13, 877–918. Bielski, B.H.J., Cabelli, D.E., Arudi, R.L., Ross, A.B., 1985. Reactivity of HO2/O2- radicals in aqueous solution. J. Phys. Chem. Ref. Data 14, 1041–1100. Buxton, G.V., Greenstock, C.L., Helrnan, W.P., Ross, A.B., 1988. Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J. Phys. Chem. Ref. Data 17, 513–886. Buxton, G.V., Stuart, C.R., 1995. Re-evaluation of the thiocyanate dosimeter for pulseradiolysis. J. Chem. Soc. Faraday Trans. 91, 279–281. Cabelli, D.E., Bielski, B.H.J., 1984a. Pulse radiolysis study of the kinetics and mechanisms of the reactions between manganese(II) complexes and HO2/O2- radicals. 1. Sulfate, formate, and pyrophosphate complexes. J. Phys. Chem. 88, 3111–3115. Cabelli, D.E., Bielski, B.H.J., 1984b. Pulse radiolysis study of the kinetics and mechanisms of the reactions between manganese(II) complexes and HO2/ O2- radicals. 2. The phosphate complex and an overview. J. Phys. Chem. 88, 6291–6294. Duncan, C., White, A.R., 2012. Copper complexes as therapeutic agents. Metallomics 4, 127–138. Durot, S., Lambert, F., Renault, J.-P., Policar, C., 2005. A pulse radiolysis study of superoxide radical catalytic dismutation by a manganese(II) complex with a Ntripodal ligand. Eur. J. Inorg. Chem. 2005, 2789–2793 (and references therein). Fielden, E.M., Roberts, P.B., Bray, R.C., Lowe, D.J., Mautner, G.N., 1974. The Mechanism of action of superoxide dismutase from pulse radiolysis and electron paramagnetic resonance. Evidence that only half the active sites function in catalysis. Biochem. J. 139, 49–60. Halliwell, B., Gutteridge, J.M.C., 2009. Free Radicals in Biology and Medicine. Oxford University Press, New York. Holm, R.H., Kennepohl, P., Solomon, E.I., 1996. Structural and functional aspects of metal sites in biology. Chem. Rev. 96, 2239–2314. Hydration enthalpies of selected ions, RSC Data Book, 〈http://www.rsc.org/Education/ Teachers/Resources/Databook/ds_hydration_enthalpies.htm〉 (accessed 10 January 2017). Iranzo, O., 2011. Manganese complexes displaying superoxide dismutase activity: a balance between different factors. Bioorg. Chem. 39, 73–87. Jacobsen, F., Holcman, J., Sehested, K., 1997. Manganese(II)−superoxide complex in aqueous solution. J. Phys. Chem. A. 101, 1324–1328.

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