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. 2022 Oct 25;119(43):e2212343119.
doi: 10.1073/pnas.2212343119. Epub 2022 Oct 13.

The photoprotection mechanism in the black-brown pigment eumelanin

Aleksandra Ilina  1   2   3 , Karen E Thorn  1   2 , Paul A Hume  1   2 , Isabella Wagner  1   2 , Ronnie R Tamming  1   2   4 , Joshua J Sutton  2   5 , Keith C Gordon  2   5 , Sarah K Andreassend  1 , Kai Chen  1   2   4 , Justin M Hodgkiss  1   2
Affiliations

The photoprotection mechanism in the black-brown pigment eumelanin

Aleksandra Ilina et al. Proc Natl Acad Sci U S A. .

Abstract

The natural black-brown pigment eumelanin protects humans from high-energy UV photons by absorbing and rapidly dissipating their energy before proteins and DNA are damaged. The extremely weak fluorescence of eumelanin points toward nonradiative relaxation on the timescale of picoseconds or shorter. However, the extreme chemical and physical complexity of eumelanin masks its photoprotection mechanism. We sought to determine the electronic and structural relaxation pathways in eumelanin using three complementary ultrafast optical spectroscopy methods: fluorescence, transient absorption, and stimulated Raman spectroscopies. We show that photoexcitation of chromophores across the UV-visible spectrum rapidly generates a distribution of visible excitation energies via ultrafast internal conversion among neighboring coupled chromophores, and then all these excitations relax on a timescale of ∼4 ps without transferring their energy to other chromophores. Moreover, these picosecond dynamics are shared by the monomeric building block, 5,6-dihydroxyindole-2-carboxylic acid. Through a series of solvent and pH-dependent measurements complemented by quantum chemical modeling, we show that these ultrafast dynamics are consistent with the partial excited-state proton transfer from the catechol hydroxy groups to the solvent. The use of this multispectroscopic approach allows the minimal functional unit in eumelanin and the role of exciton coupling and excited-state proton transfer to be determined, and ultimately reveals the mechanism of photoprotection in eumelanin. This knowledge has potential for use in the design of new soft optical components and organic sunscreens.

Keywords: eumelanin; excited-state proton transfer; excitons; photochemistry; ultrafast spectroscopy.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Eumelanin as a heterogenous ensemble of chromophores. (A) Molecular structures of eumelanin building blocks, DHI and DHICA. (B) Schematic depiction of eumelanin structure, comprising oligomers stacked in aggregates and larger particles. (C) Measured UV–visible absorption spectrum of a thin film of synthetic eumelanin, along with illustration of underlying chromophore spectra that could sum to give the measured spectrum. (D) Wavelength-dependent resonance Raman spectra of a thin film of synthetic eumelanin is presented for 11 Raman pump excitation wavelengths across a 351- to 647-nm range under the same experimental conditions. We observe here a peak shift in the dominant ground-state Raman band peak decreasing from ∼1,605 cm−1 to ∼1,575 cm−1 with decreasing excitation energy.
Fig. 2.
Fig. 2.
Distribution of chromophores excited in eumelanin films, shown by ultrafast TA spectroscopy and modeling. (A–E) TA spectral dynamics for synthetic eumelanin films with a variety of different excitation energies indicated. In each case, an absence of spectral dynamics is observed on the picosecond timescale, excluding the possibility of dynamic energy transfer within the ensemble of chromophores. Each set of spectra features a photoinduced absorption peak around 1.3 eV, with a higher-energy tail elongated toward the position of the ground-state bleach at the excitation wavelength. (F–H) TA spectra are modeled as a series of overlapping basis functions in a similar way as the absorption spectrum is fit in Fig. 1, but using bisignate curves as the TA basis spectra. The basis spectra (with one basis spectrum highlighted in bold to show the function shape) are shown in F, with comparison between experimental and fitted TA spectra in G, and spectra of fitted coefficients for each excitation wavelength in H. Further modeling details are provided in SI Appendix.
Fig. 3.
Fig. 3.
Fluence-dependent TA signals. TA kinetics traces of the main photoinduced absorption (PIA) peak at 1.3 eV for the excitation energy of 2.3 eV. (Inset) Linear dependence of the initial kinetics amplitude on the excitation fluence.
Fig. 4.
Fig. 4.
Comparison of the kinetics and spectral shapes of eumelanin and DHICA monomer. Both TA (A) and PL (B) kinetics of DHICA monomer (in neutral water) and a eumelanin film demonstrate similar excited-state decay rates. (Insets) The spectral shapes for the different experiments.
Fig. 5.
Fig. 5.
ESPT in DHICA. (A) Four-level system demonstrating the proposed ESPT energy dissipation pathway in DHICA monomer in neutral environment. (B) Steady-state absorption (dashed line) and emission (solid line) spectra of the four studied forms of DHICA monomer. (C) Excited-state decay of these forms.
Fig. 6.
Fig. 6.
FSRS of ESPT in DHICA and eumelanin. (A) FSRS spectral series for DHICA in neutral water across a 400-fs to 10-ps time range. (B) FSRS spectral series across the same time range for a eumelanin film. The distinctive shift over time from higher to lower wavenumber of the peak around the 1,600 cm−1 Raman band is observed for both eumelanin and DHICA. (C and D) Time-dependent shift of the peak near 1,600 cm−1 for DHICA and eumelanin. Dashed horizontal lines indicate the frequencies of relevant modes for excited-state protonated (catechol) and deprotonated (catecholate) forms of DHICA calculated by DFT. (E) Corresponding vibrational modes from DFT calculations.
Fig. 7.
Fig. 7.
Schematic depiction of the two-step relaxation model elucidated for eumelanin: First, ultrafast internal conversion among coupled localized exciton states produces a lower energy distribution of excitations within 100 fs, then excited-state proton transfer to the solvent (and proton recapture) proceeds in ∼4 ps.
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