Stability and Catalase-Like Activity of a Mononuclear Non-Heme Oxoiron(IV) Complex in Aqueous Solution

Heme-type catalase is a class of oxidoreductase enzymes responsible for the biological defense against oxidative damage of cellular components caused by hydrogen peroxide, where metal-oxo species are proposed as reactive intermediates. To get more insight into the mechanism of this curious reaction a non-heme structural and functional model was carried out by the use of a mononuclear complex [FeII(N4Py*)(CH3CN)](CF3SO3)2 (N4Py* = N,N-bis(2-pyridylmethyl)- 1,2-di(2-pyridyl)ethylamine) as a catalyst, where the possible reactive intermediates, high-valent FeIV=O and FeIII–OOH are known and spectroscopically well characterized. The kinetics of the dismutation of H2O2 into O2 and H2O was investigated in buffered water, where the reactivity of the catalyst was markedly influenced by the pH, and it revealed Michaelis–Menten behavior with KM = 1.39 M, kcat = 33 s−1 and k2(kcat/KM) = 23.9 M−1s−1 at pH 9.5. A mononuclear [(N4Py)FeIV=O]2+ as a possible intermediate was also prepared, and the pH dependence of its stability and reactivity in aqueous solution against H2O2 was also investigated. Based on detailed kinetic, and mechanistic studies (pH dependence, solvent isotope effect (SIE) of 6.2 and the saturation kinetics for the initial rates versus the H2O2 concentration with KM = 18 mM) lead to the conclusion that the rate-determining step in these reactions above involves hydrogen-atom transfer between the iron-bound substrate and the Fe(IV)-oxo species.


Introduction
Superoxide dismutases (SODs), catalase-peroxidases (KatGs) and catalases are specialized oxidoreductase enzymes for the degradation of reactive oxygen species (ROS), e.g., hydrogen peroxide, hydroxyl and superoxide radicals to avoid their accumulation and prevent the oxidative damage of cellular components, that may lead to a number of diseases such as cancer, Alzheimer's diseases and aging [1][2][3][4]. For example, the hydroxyl and/or hydroperoxyl radicals may cause lipid peroxidation, membrane damage, DNA oxidation and cell death [5,6]. As a fine coupling of SODs and catalases, the former enzymes catalyze the dismutation of superoxide into dioxygen (1-electon oxidation) and H 2 O 2 , whilst the latter enzymes eliminate the H 2 O 2 via its decomposition by disproportionation into O 2 (2-electron oxidation) and H 2 O, resulting in the optimal intracellular concentration of a H 2 O 2 molecule [7][8][9], which acts as a second messenger in signal-transduction pathways. Otherwise, it is worth to note, that the therapeutic potential of H 2 O 2 makes this molecule also a valuable target in cancer killing via chemo-and radiotherapy, and in stroke therapy [10][11][12].

Catalase-Like Reactivity of [Fe II (N 4 Py*)(CH 3 CN)](CF 3 SO 3 ) 2 in Aqueous Solution
The catalase-like activity of the complex [Fe II (N 4 Py*)(CH 3 CN)](CF 3 SO 3 ) 2 to disproportionate H 2 O 2 into H 2 O and O 2 was investigated in aqueous solution at 20 • C by gasvolumetric measurements of evolved dioxygen. To gain further information on the mechanism of catalase activity of our iron complex, we first examined pH-dependence of catalase activity. It was reported that the coordination and dissociation of peroxides on metal-porphyrins are pH dependent reactions [47,48]. Moreover, they reported that the coordination is accelerated at a higher pH region and that the subsequent O-O bond cleavage leading to the formation of high-valent oxo-Fe(IV) or oxo-Fe(V) species is pH-independent (only at higher pH region, where the protonation of the distal oxygen in the peroxo-complex can be excluded) irreversible reaction. These results suggest that the coordination of peroxides is a crucial step for the formation of high-valent Fe species, and the mechanism of catalase activity involves the coordination of H 2 O 2 , which is considered to be pH-dependent as well. Therefore, we hypothesized that formation of reactive intermediate 2 is accelerated at pH 9.5 and catalase activity is increased as compared at pH 8. As shown in Figure 1, O 2 production of 1 in 50 mM borate buffer (pH 9.5) was significantly higher than that in phosphate buffer (pH 8). V in value under this condition was determined to be V in = 1.13 × 10 −3 Ms −1 , which is approximately seven times higher than that at pH 8, and 8.5 times higher than that at pH 11. This indicates that the rate-determining step was faster at pH 9.5 than at pH 8, which may be explained by the higher concentration of the more nucleophilic HO 2 -.
The pH dependence of H 2 O 2 dismutation was further studied between pH 7 and pH 11. It was found that the initial rate of the disproportionation of H 2 O 2 increases with increasing pH and goes through a maximum. The pH profile of 1 exhibits a sharp optimum at pH~9.5, whereas catalases in general exhibit a broad pH optimum extending from pH 5.6 to 8.5 [48]. In control experiments, in the absence of the complex, the pH of the solution did not change in the presence of H 2 O 2 , and no significant O 2 volume was evolved. We believe that the activity is influenced by the protonation state of H 2 O 2 . Assuming that hydrogen peroxide is activated by a direct interaction with the Fe IV =O group of the complex, decomposition is expected to be favored by a high pH because of the larger concentration of the hydroperoxide anion (HOO − is more nucleophilic than H 2 O 2 ). On the other hand, at higher pH values, the complex may be destroyed by the formation of the mineral forms of iron or catalytically inactive, insoluble µ-oxo-diiron(III) species.
Detailed kinetic studies on the disproportionation of H 2 O 2 were performed in aqueous solution (pH 9. The data presented illustrate that the catalyst had a relatively high turnover number (k cat ) but appeared to bind peroxide very badly. The K M value was greater than the values for the natural enzymes from Thermus thermophilus (K M = 0.083 M) [19,20], Tricholoma album (K M = 0.015 M) [21] and Lactobacillus plantarum (K M = 0.35 M) [17,18] indicating a lower affinity to the substrate. The k cat value equaled 33 s −1 , however, was 3-4 times magnitudes lower when compared to the natural enzymes Thermus thermophilus (k cat = 2.6 × 10 5 s −1 ), Tricholoma album (k cat = 2.0 × 10 5 s −1 ), Lactobacillus plantarum (k cat = 2.6 × 10 4 s −1 ) and the heme-containing catalases (k cat = 4 × 10 7 s −1 ). Despite this iron complex presents lower values of catalytic efficiency than other models (Table 1) [49][50][51][52], it must be emphasized that this value was obtained in water and in pH close to the natural, representing an advantage of the title complex with respect to most of the published models, whose studies have been conducted in organic solvent due to the lack of solubility or activity in aqueous solution.

Catalase-Like Reactivity Mediated by [(N4Py*)Fe IV =O](ClO 4 ) 2 in Aqueous Solution
Rohde and co-workers have shown that the independently prepared [(N4Py)Fe IV =O] 2+ reacts rapidly with near-stoichiometric H 2 O 2 resulting in dioxygen and [Fe II (N4Py)(CH 3 CN)] 2+ in acetonitrile [54]. Later Browne and co-workers have found clear evidence for the reaction of Fe III -OOH with H 2 O 2 in methanol [55]. In their case the oxoiron(IV) intermediate can also be formed by homolytic cleavage of the O-O bond of an Fe III -OOH, but the rate of its formation is much lower than the Fe III -OOH-mediated H 2 O 2 disproportionation observed with high excess H 2 O 2 under catalytic conditions. As a continuity of these studies, we attempted to directly investigate the reactivity of the possible intermediates (Fe IV =O, Fe III -OOH) during the catalase reaction in aqueous solution.
We have shown earlier that complex 1 forms very stable high valent oxoiron(IV) species (2) with PhIO in CH 3 CN (t 1/2 = 233 h at R.T., λ max = 705 nm, ε = 400 M −1 cm −1 ) [43]. As a test of our oxoiron(IV) species we firstly investigated its reaction with excess H 2 O 2 (75 equiv.) in acetonitrile at 10 • C, which resulted in the formation of a relatively stable transient purple species with a characteristic absorbance maximum at λ max 535 nm (ε = 1100 M −1 cm −1 ; Figure 2a). It had a half-life of about 3 min even at 25 • C, but its decay can be remarkably enhanced by the addition of H 2 O into the Fe III -OOH-containing solution (CH 3 CN/H 2 O = 1:1) with a k obs value of about 12.3 × 10 −3 s −1 at 10 • C, resulting in the formation of 2 (Figure 2b). It is worth to note that at higher pH the decay was so fast, that we were not able to follow it. These results might suggest that a high-valent oxoiron(IV) species was one of the possible intermediates that may be responsible for the dismutation of H 2 O 2 in aqueous solution. In the iron-catalyzed oxidation of H 2 O 2 with terminal oxidants four processes can be proposed as the rate-controlling step, namely the formation of Fe III -OOH or high-valent oxoiron(IV), or their reaction with the substrate (H 2 O 2 ). To avoid this difficulty, and to get more insight into the mechanism of the H 2 O 2 oxidation process we synthesized the oxoiron(IV) complex 2 by an in situ reaction of 1 with PhIO in acetonitrile, and investigated its stability and reactivity with H 2 O 2 in a buffered H 2 O-CH 3 CN mixture (v/v = 1:1). In this way the role of the oxoiron(IV) species could be directly investigated. The UV-vis spectra of 2 in buffered solutions were almost identical to that observed in the acetonitrile. The observed blue shift on the λ max values (from 705 to 697 nm) might be explained by the interaction (H-bridge) of the oxoiron(IV) with the H 2 O molecule(s).
The reactivity of 2 was found to depend significantly on the pH value of reaction solutions. The maximum rate of H 2 O 2 dismutation, k' obs (k' obs = k obs − k sd from the −d [2]/dt = k obs [2] = (k sd + k' obs ) [2]) could be observed at pH 9, where the self decay process (k sd ) could be neglected (Figure 4a). The increase of the k obs at higher pH could be explained by the self decay of 2. Addition of 10 equiv.   Detailed kinetic and mechanistic studies were carried out in buffered water/acetonitril mixture (v/v = 1:1) in pH 8, close to the natural at 10 • C, where the self decay process can be excluded. The reactivity of 2 was monitored by UV-vis spectroscopy and the rate of its rapid decomposition was measured at 697 nm (Figure 5a). Pseudo-first order fitting of the kinetic data allowed us to determine k obs values. These results indicate a direct reaction between 2 and H 2 O 2 . In order to investigate the possible involvement of a hydrogen atom in the rate-determining step we investigated the reactivity of 2 with H 2 O 2 in buffered MeCN/D 2 O/H 2 O (v/v = 1:0.75:0.25). Solutions of 2 in the presence of D 2 O at pH 8 were somewhat less reactive against H 2 O 2 , yielding a solvent kinetic isotope effect of 6.2. This value was significantly smaller than that was obtained for the H-D isotope effect for [Ru IV O(bpy) 2 (py)] at pH 2.3 (KIE = 22.1 ± 1.2), but almost identical with that was measured at pH 9.7 (KIE = 8 ± 2.9) at 25 • C [40]. The most straightforward interpretation of the proton dependence was that the pathways involve the acid-base pre-equilibrium of
Catalytic reactions were carried out at 20

Conclusions
It was found earlier that non-heme oxoiron(IV) complexes were able to carry out electrophilic Based on detailed mechanistic studies on H 2 O 2 oxidation that were investigated with in situ generated oxoiron(IV) species, plausible mechanisms were proposed, in which the H 2 O 2 oxidation occurred by the HAT mechanism. To put together the stoichiometric and catalytic results it could be said that the highest catalytic activity of the H 2 O 2 dismutation could be observed at pH 9.5, where the concentration of the more nucleophilic hydroperoxide anion (HOO − ) was high, and the self-decay of the oxoiron(IV) intermediate could be neglected. These results were in good agreement with the electrophilic reactivity of oxoiron(IV) intermediates proposed for heme-type monoiron catalases, and might help us to understand the mechanism of the detoxification of H 2 O 2 in biological systems.
Author Contributions: Individual contribution of authors were as follows: B.K., Organic synthesis; B.S., Reaction kinetics; G.S., Senior supervisor and advisor; and J.K., Project leader, writer of the manuscript.
Funding: This research received no external funding.