A XAS Study of the Sulphur Environment in Human Neuromelanin and Synthetic Analogues

Neuromelanin (NM) is a complex insoluble polymeric pigment present in neurons of different brain regions of several animal species including humans (1). NM is known to accumulate with aging, and an enhanced rate of degeneration of highly pigmented neurons has been claimed, thus linking it to development of Parkinson’s disease (PD). In PD the concentration of NM decreases rapidly due to the selective loss of pigmented dopaminergic neurons. Non-pigmented neurons are mostly conserved and it was suggested that NM, depending on cellular context, can be either protective against or rather promote the pigmented dopaminergic neurons degeneration. NM is mainly constituted by a melanic component, but the presence of both peptidic and lipidic components has been observed (1,2,3). The melanic component is composed by two classes of molecules in rather well determined proportions. One is a benzothiazine-based molecule characteristic of pheomelanin, that is formed through the incorporation of cysteine with dopamine, and makes up around 20-25% of the total melanic component of natural NM. The other one is an indole-based molecule characteristic of eumelanin that is formed through the oxidation of dopamine (4). X-Ray diffraction studies (5) have shown that NM has a multilayer (graphite-like) three-dimensional structure made of planar overlapping sheets of molecules containing indolebenzothiazine rings. It was shown by scanning probe and photoelectron emission microscopy that NM granules are comprised of spherical structures with a diameter of ~30 nm, with pheomelanin at the core and eumelanin at the surface (6). In human NM the peptidic component accounts for the 15% of the neuromelanin weight (3). Furthermore it has been found that about 3% in weight of the peptidic component is cysteine. Chemical degradation methods have suggested that sulphur (S) in NMs is present in two different binding sites, namely as cysteine and coordinated to benzothiazine-like rings. However, a direct spectroscopic demonstration of the presence of S in heterocyclic thiazine- and cysteine-like structure is not available. Furthermore previous studies (4) had already reported that chemical degradation of synthetic melanins and natural NM generates typical molecules that derive from benzothiazine rings, but a direct and non destructive demonstration of this fact is still missing. A detailed characterization of the NM structure and its interaction with other intraneuronal compounds is hence an essential step towards the understanding of the mechanism of its synthesis, interaction with other neuronal-glial compounds, its metabolic fate and the role it plays in normal aging and PD. In the present work X-ray Absorption Spectroscopy (XAS) was used to characterize the local structure around S atoms in both natural and synthetic melanins. XAS spectra in the energy region around the S absorption K-edge (the so called X-ray Absorption Near Edge Structure -XANES- region) have been collected at the D04B bending magnet beam line of the Brazilian Synchrotron Light Laboratory. We acquired spectra of 1) natural melanin isolated from Cerebellum, 2) three synthetic melanins prepared according to different procedures and 3) two S containing model compounds, namely trichochrome and cysteine. Due to its chemical selectivity and sensitivity to the local atomic arrangement, XAS is a suitable technique for structural studies on biological material aimed at characterizing the atomic structure around a given absorber. In particular, the spectral structure of the XANES region is sensitive to the electronic structure of the absorber and the symmetry of the local environment around it. Although in our case a quantitative interpretation of this part of the spectrum was not possible, a qualitative comparison of the XANES regions of spectra has given valuable information on similarities and differences between local geometries around the S in different compounds (7,8). Our analysis strategy is based on the observation that XAS spectra of samples where the absorbing atom is present in more than one structure can be obtained as the sum of appropriate normalized model spectra weighted by the fraction of absorbing atom present in each component (9). Since the structure of our model compounds is well known, the XANES spectrum of them can be used as fingerprints of well defined S atomic environments as done for other absorbing centers. From a direct comparison among the spectra (see Fig.1) of the various samples one sees that the spectral features of the two synthetic melanins and of natural melanin are quite similar. In addition, our analysis suggested the presence of heterocyclic S of the benzothiazine type in both synthetic melanins and natural NM. Finally we have proved that S in natural NM appears in two different structural coordination modes, namely either bound to benzothiazine-like rings or present in the cysteine residue. Figure 1. Spectra of synthetic (DAC, DEC, Pheomelanin) and natural (HNM) melanins. This Figure is shown in the abstract book. REFERENCES 1. Zucca, F. A., G. Giaveri, M. Gallorini, A. Alberini, M. Toscani, G. Pezzoli, R. Lucius, H. Wilms, D. Sulzer, S. Ito, K. Wakamatsu, and L. Zecca. 2004. Pigment Cell Res. 17:610-617. 2. Zecca, L., C. Mecacci, R. Seraglia, and E. Parati. 1992. Biochim. Biophys. Acta. 1138:6–10. 3. Zecca, L., P. Costi, C. Mecacci, S. Ito, M. Terreni, S. Sonnino. 2000. J. Neurochem. 74:1758-1765. 4. Wakamatsu, K., K. Fujikawa, F. A. Zucca, L. Zecca, and S. Ito. 2003. J. Neurochem. 86:1015-1023. 5. Crippa, P. R., Q. J. Wang, M. Eisner, S. C. Moss, L. 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