The photoreactive free radical in eumelanin

Abstract

Melanin is the primary photoprotecting pigment in humans as well as being implicated in the development of deadly melanoma. The material also conducts electricity and has thus become a bioelectronic model for proton-to-electron transduction. Central to these phenomena are its spin properties-notably two linked species derived from carbon-centered and semiquinone radicals. Using a novel in situ photoinduced electron paramagnetic resonance technique with simultaneous electrical measurements, we have elucidated for the first time the distinct photoreactivity of the two different radical species. We find that the production of the semiquinone is light- and water-driven, explaining the electrical properties and revealing biologically relevant photoreactivity.

Fig. 1. Comproportionation equilibrium.

Indolequinone moieties of different oxidative states react with water to form hydronium and an intermediate state, the SQR. The light-activated and dark mechanisms are summarized in the brackets. With light-activated reactions, light induces a redox reaction, followed by dissociation. For dark reactions, dissociation occurs first, followed by a redox reaction. For more details, see fig. S1 and Supplementary Text.

Fig. 2. Melanin X-band EPR spectrum obtained under vacuum (that is, dry).

The black and orange curves are at microwave powers of 2 and 585 mW, respectively. The purple curve (585 mW) was obtained after exposure to ammonia vapor and dried under vacuum. It can be seen that the peak at g = 2.0049 is reduced with increasing microwave power, whereas a peak at g = 2.0075 emerges. The addition of base increases the relative strength of the g = 2.0075 peak, thus showing that the strength of the SQ free radical signal (g = 2.0075) can be enhanced vis-à-vis the carbon-centered free radical signal (g = 2.0049). Uncertainties shown indicate the noise in the baseline. a.u., arbitrary units.

Fig. 3. Photo-EPR time traces for neutral melanin.

(A) Photo-EPR time traces at 0.063-mW microwave power and monitored at g = 2.0049, the CCR feature. The colors indicate hydration state, which was obtained by normalizing the vapor pressure to a previously published adsorption isotherm (33): 0% (black), 11.6% (blue), and 17.2% (red) weight gain in water relative to melanin dry mass. Traces have been offset from one another for clarity. Dashed lines indicate baseline before the light is switched on. (B) Photo-EPR time trace at 199.2 mW and positioned at g = 2.0075, the SQR feature. Data point markers indicate the mean and uncertainties in the baseline (squares) and the signal (circles).

Fig. 4. Representative photo-EPR time trace (optimized for the SQR, 11.6% weight gain in water content) overlaid on a photocurrent time trace obtained for a contacted melanin sample and obtained simultaneously in situ.

The data confirm that the SQ free radical is explicitly linked to the conductivity and hence the protonic conducting behavior of solid-state melanin. Data point markers indicate the mean and uncertainties in the initial baseline (square), the signal (circle), and the final baseline (triangle).