Biochimica et Biophysica Acta (BBA) - General Subjects
Mechanism of dopachrome tautomerization into 5,6-dihydroxyindole-2-carboxylic acid catalyzed by Cu(II) based on quantum chemical calculations
Introduction
Melanins, one of the most important pigments in animals, are known to have various functions that are essential for living organisms. The biosyntheses of melanins mainly occur in the melanosomes, membrane bound organelles within pigment cells such as melanocytes which are distributed in the epidermis, the hair follicle, the inner ear, and the eye [1], [2], [3], [4]. Biosynthesized melanins consist of two distinct pigments, black to brown eumelanins and yellow to reddish-brown pheomelanins [5], [6], [7], [8], [9], [10]. Especially, the photoprotective and antioxidative effects of eumelanins have extensively been investigated as major functions although the high photosensitizing effects of pheomelanins have been considered to confer cytotoxicity [11]. Neuromelanins, melanin-like pigments which are mainly found in the neurons of substantia nigra and locus coeruleus, have also been recognized to play important physiological roles such as sequestration of heavy metal ions and methylphenylpyridine (MPP+) which is known to cause the Parkinson's disease [12], [13]. Although the biological and medical relevance of melanins and their biosyntheses have been subject to controversy [14], [15], it is well accepted that eumelanins have an ability to convert absorbed light energy into heat energy [16], [17], [18] and to detoxify reactive oxygen species (ROS) [19], [20], [21].
Eumelanins are basically recognized as stacked and aggregated oligomers or large heteropolymers that are mainly composed of two monomers, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [22], [23], [24]. The biosynthesis of DHICA is achieved by tautomerization of dopachrome, an intermediate in melanogenesis (Fig. 1) [25], [26]. Dopachrome is a comparatively unstable molecule that slowly converts to DHI via decarboxylation without any enzymes at physiologically usual pH (Fig. 1) [27], [28]. Due to this nature of dopachrome, eumelanin had been initially considered to consist mostly of DHI in early research on melanin chemistry. However, this view was reconsidered by analyses mainly based on chemical degradation that natural eumelanins include DHI and DHICA units at a nearly equal ratio [29].
In an experiment, it was demonstrated that DHICA units in eumelanin are responsible for the antioxidative properties of the pigment [30]. As well as eumelanin itself, DHICA and its methoxy metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA) were also found to exhibit considerable antioxidant activity [31]. Recently, it was also reported that DHICA affects cross-talk among epidermal cells, implying the pivotal roles of this molecule in skin homeostasis and cell-protection [32]. These evidences strongly show the physiological significance of DHICA metabolism.
Experimental works have found some key factors to tautomerize to DHICA; divalent metal ions, especially Cu(II) [33], [34], [35], and dopachrome tautomerase (DCT), which contains a pair of Zn(II) ions at its active sites [36], [37], [38], can strongly catalyze the tautomerization of dopachrome. Although the effect of Zn(II) itself on the tautomerization has shown to be considerably weaker than that of Cu(II) [33], DCT is significantly potent in catalyzing the reaction. A comparative study of the tautomerization effect between DCT and Cu(II) revealed that DCT is more intensive to catalyze the tautomerization than Cu(II) [39]. This result may give us an impression that the effect of Cu(II) is not a major factor for DHICA formation. However, although DCT expression in human follicular melanocytes from elderly individuals had been shown to be scarcely detectable [40], chemical degradation analyses clarified that these samples contain a relatively high ratio of DHICA (33–45%) [41], [42]. Therefore, a reappraisal of the contribution of Cu(II) towards the DHICA formation might afford a new understanding of melanin synthesis.
Experimental findings mentioned above trigger us to ask further question about the mechanisms of how these factors selectively tautomerize dopachrome. Previously, we clarified the mechanism of dopachrome conversion towards DHI formation based on the results obtained from first-principles calculation [43]. In this connection, the mechanistic difference of DHI/DHICA formation deserves attentions as a means of obtaining the generalized view of dopachrome conversion. A proposed mechanism of the DCT-catalyzed reaction may clearly explain the selectivity towards the formation of DHICA over DHI. According to the strong stereospecificity of DCT, an involvement of an interaction between carboxylate group of dopachrome and an unidentified group of DCT in the catalysis of DCT has been suggested as well as the participation of Zn(II) ions [38]. In this picture, the selective formation of DHICA will be described in terms of inhibition of decarboxylation, that leads to the reduction of the DHI formation. However, the clear pictures of the metal ions-catalyzed conversions have been poorly established despite the biological importance. Although metal ions are supposed to exert their catalytic activities to the tautomerization by interacting with quinonoid oxygens (5,6-oxygens, see Fig. 1) of dopachrome [33], the reason why such interactions could realize the selective formation of DHICA is unclear.
To obtain the clear-cut view of the formation of DHICA catalyzed by Cu(II), we consider a paired Cu(II)-dopachrome interacting system as a minimal model that would result in exclusive DHICA formation from dopachrome. Since the metal ions-catalyzed reactions are known to proceed by first-order kinetics [33], this modeling is consistent with the relevant experimental systems. Using this model, we present a theoretical evaluation of dopachrome conversion catalyzed by Cu(II). To gain a reliable description based on universally defined energy profile along the reaction pathway, we employ a density functional theory-based calculation.
Dopachrome conversion proceeds via three possible dissociations; α-deprotonation, β-deprotonation, and decarboxylation (Fig. 1). As can be understood by referencing the structures of the products, decarboxylation and α-deprotonation are respectively associated with the formation of DHI and DHICA. β-Deprotonation is accompanied with a formation of quinone methide intermediate (Fig. 2). From our calculations, we here show significant reductions of the activation barriers of α-deprotonation, β-deprotonation, and decarboxylation from dopachrome by coordination of Cu(II) to quinonoid oxygens (5,6-oxygens) of dopachrome, with the lowest activation barrier of β-deprotonation among them. As demonstrated in our previous work [43], β-deprotonation and quinonoid protonation (O5/O6-protonation) are important to form DHI. However, our results obtained in this work showed that the Cu(II) coordination to quinonoid oxygens inhibits the quinonoid protonation, leading to the preference of proton rearrangement from β-carbon to carboxylate group but not to the quinonoid oxygens. Integrating these results, we conclude that dopachrome tautomerization first proceeds via proton rearrangement from β-carbon to the carboxylate group and subsequently undergoes α-deprotonation to form DHICA.
Section snippets
Density functional theory-based calculation
In this work, we performed first-principles calculations based on density functional theory [44], [45] with the Becke's three-parameter hybrid functional [46] combined with the Lee–Yang–Parr correlation functionals [47] (B3LYP). Calculations were carried out with 6-31++G(d,p) basis set using the Gaussian 09 suite of programs [48].
Solvation model
To appropriately reflect the stabilization by the dielectric response of surrounding water molecules, we use the integral equation formalism polarizable continuum
Effect of Cu(II) on activation energies of α-deprotonation, β-deprotonation, and decarboxylation
First, we evaluate the activation energies of α-deprotonation, β-deprotonation, and decarboxylation from dopachrome in the presence of coordination of Cu(II) to quinonoid group. As described in the Introduction section, α-deprotonation, β-deprotonation, and decarboxylation are respectively associated with the formation of DHICA, quinone methide intermediate, and DHI. Thus, the comparison of activation energies of these dissociations will provide an essential information to understand the
Conclusions
In this paper, we describe the mechanism of dopachrome tautomerization towards DHICA catalyzed by Cu(II) in aqueous solution. Despite the biological significance of this reaction and the existence of experimental data suggesting the strong catalytic behavior of Cu(II), the reaction mechanism of dopachrome tautomerization in the presence of Cu(II) has not been clarified. Our thoroughly examined model, in which Cu(II) is coordinated to quinonoid oxygens (5,6-oxygens) of dopachrome and two water
Acknowledgements
This work is supported in part by:
MEXT Grant-in-Aid for Scientific Research on Innovative Areas Program (2203–22104008) and Scientific Research (A) (26248006); JST ALCA Program “Development of Novel Metal-Air Secondary Battery Based on Fast Oxide Ion Conductor Nano Thickness Film”; JSPS Core-to-Core Program “A. Advanced Research Networks: Computational Materials Design on Green Energy”; and the Osaka University Joining and Welding Research Institute Cooperative Research Program.
Some of the
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