The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface

Abstract

Neuromelanin (NM) isolated from the substantia nigra region of the human brain was studied by scanning probe and photoelectron emission microscopies. Atomic force microscopy reveals that NM granules are comprised of spherical structures with a diameter of approximately 30 nm, similar to that observed for Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected at specific wavelengths of UV light between 248 and 413 nm, using the spontaneous-emission output from the Duke OK-4 free electron laser. Analysis of the data establishes a threshold photoionization potential for NM of 4.5 +/- 0.2 eV, which corresponds to an oxidation potential of -0.1 +/- 0.2 V vs. the normal hydrogen electrode (NHE). The oxidation potential of NM is within experimental error of the oxidation potential measured for human eumelanosomes (-0.2 +/- 0.2 V vs. NHE), despite the presence of a significant fraction of the red pigment, pheomelanin, which is characterized by a higher oxidation potential (+0.5 +/- 0.2 V vs. NHE). Published kinetic studies on the early chemical steps of melanogenesis show that in the case of pigments containing a mixture of pheomelanin and eumelanin, of which NM is an example, pheomelanin formation occurs first with eumelanin formation predominantly occurring only after cysteine levels are depleted. Such a kinetic model would predict a structural motif with pheomelanin at the core and eumelanin at the surface, which is consistent with the measured surface oxidation potential of the approximately 30-nm constituents of NM granules.

Fig. 1.

SEM image of dried NM pigment isolated from the SN region of human brain tissue. The image illustrates a commonly observed, large, amorphous aggregate (>10 μm in size) composed of smaller NM molecules, roughly spherical in shape and varying widely (200–500 nm) in diameter. The image is 6.75 μm × 4.50 μm.

Fig. 2.

AFM images of SN NM. The small substructures of NM, ≈30 nm in diameter, are observed both in isolation [A (height) and B (phase)] and in the highly aggregated structures [C (phase)]. The images in A and B are 950 nm × 950 nm; the image in C is 1.57 μm × 1.57 μm.

Fig. 3.

PEEM image of isolated (A) and aggregated (B) NM upon UV excitation by an FEL at 248 nm, and wavelength-dependent FEL-PEEM data plotted and fit according to Eq. 1 (C). The best fit to the experimental data gives a photoionization threshold of 4.5 ± 0.2 eV (275 nm) for both isolated and aggregated deposits. Data are shown for the isolated granule. The image in A is 2.3 μm × 2.3 μm; the image in B is 1.1 μm × 1.5 μm.