Spectroelectrochemical Reverse Engineering DemonstratesThat

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Spectroelectrochemical Reverse Engineering Demonstrates that Melanin’s Redox and Radical Scavenging Activities Are Linked Eunkyoung Kim, Mijeong Kang, Tanya Tschirhart, Mackenzie Malo, Ekaterina Dadachova, Gaojuan Cao, Jun-Jie Yin, William E Bentley, Zheng Wang, and Gregory F. Payne Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01166 • Publication Date (Web): 15 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Spectroelectrochemical reverse engineering demonstrates that melanin’s redox and radical scavenging activities are linked Eunkyoung Kim,†‡ Mijeong Kang, †‡ Tanya Tschirhart,§ Mackenzie Malo,¶ Ekaterina Dadachova,¶ Gaojuan Cao,# Jun-Jie Yin,# William E. Bentley,†‡ Zheng Wang,⊥ Gregory F. Payne*†‡

†

Institute for Bioscience and Biotechnology Research, University of Maryland, 4291 Fieldhouse Drive, 5112 Plant Sciences Building, College Park, Maryland 20742, USA

‡

Fischell Department of Bioengineering, University of Maryland, 8228 Paint Branch Drive, 2330 Jeong H. Kim Engineering Building, College Park, Maryland 20742, USA §

American Society for Engineering Education, Postdoctoral Fellowship Program, US Naval Research Laboratory, Washington, D.C. 20375, USA ¶

College of Pharmacy & Nutrition, University of Saskatchewan, Canada

#

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, United States 1

ACS Paragon Plus Environment

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⊥

Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, DC, 20375, USA

KEY WORDS Fungal melanin, Spectroelectrochemistry, Reverse engineering, Redox activity, Radical scavenging

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ABSTRACT Melanins are ubiquitous in nature but their biological activities and functions have been difficult to discern. Conventional approaches to determine material function start by resolving structure and then characterize relevant properties.

These approaches have been less successful for

melanins because of their complex structure and insolubility, and because their relevant properties are not readily characterized by conventional methods. Here, we report a novel spectroelectrochemical reverse engineering approach that focuses on redox and radical scavenging activities. In this method, the melanin is immobilized in a permeable hydrogel film adjacent to an electrode and this immobilized melanin is probed using diffusible mediators and complex electrical inputs.

Response characteristics are measured using two modalities,

electrochemical currents associated with the reaction of diffusible mediators, and optical absorbance associated with the presence of diffusible free radicals. Using this method, we observed that both Sepia and fungal melanins are redox active and can repeatedly exchange electrons to be switched between oxidized and reduced states. Further, we observed that these melanins can quench radicals either by donating or accepting electrons. Finally, we demonstrate that the melanins’ radical scavenging activities are dependent on their redox state such that a melanin must be reduced to have donatable electrons to quench oxidative free radicals, or must be oxidized to accept electrons from reductive free radicals. While the observation that melanin is redox-active is consistent with their well-accepted beneficial (radical-scavenging) and detrimental (pro-oxidant) activities, these observations may also support less well-accepted proposed functions for melanin in energy harvesting and redox communication.

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INTRODUCTION Melanins are ubiquitous in nature and produced by organisms that include single-celled microbes, plants and animals.1-4 While the biological functions of melanins are not fully resolved, they are often proposed to serve protective functions3,

5-7

and especially protection

against adverse environmental insults as illustrated in Scheme 1a1,

8-10

. Probably, the most

extreme example is the observation that black melanized fungi can survive the highly radioactive environment of the damaged Chernobyl nuclear reactor.1, 11 Melanins are also proposed to play important roles in host-pathogen interactions. Scheme 1a illustrates that fungal pathogens are believed to use cell wall bound melanins to neutralize the reactive oxygen species generated by the host’s immune cells.9,

12-14

In contrast, insects generate melanin as part of their innate

immune response and appear to use this melanin to encapsulate invading pathogens.15-17 Although there is general agreement that melanins perform protective functions in biology, a detailed mechanistic understanding is often lacking.13,

18, 19

The conventional approach to

investigate the function of biological materials typically begins by clarifying structure (e.g., repeating unit and hierarchical structure) followed by characterization of relevant properties. This conventional approach has been difficult to apply to melanins because their structures are complex and not readily resolved using traditional physical-chemical methods. Further, the properties that are generally believed to be relevant for melanin’s protective functions are redox and radical scavenging activities which are not readily assessed through common methods. Here, we characterize the properties of melanin using an alternative top-down and systemslevel reverse engineering approach.20-22 Using recently-developed electrochemical reverse engineering methods to investigate melanin’s redox properties, we previously showed that both 4


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tyrosine-derived eumelanin and tyrosine-cysteine-derived pheomelanin are redox-active and can be repeatedly oxidized and reduced.23-27 Here, we extend this methodology to spectroelectrochemical reverse engineering to allow a simultaneous probing of redox and radical scavenging activities, and we especially focus on the fungal 1,8-dihydroxynaphthalene (DHN)melanin, which is associated with pathogenesis and resistance to environmental stresses in fungi.28-31 Specifically, we studied "ghosts" of melanized fungal cells which are prepared by removing the internal cell components and leaving behind an outer melanin-rich shell associated with the cell wall.29, 32 Scheme 1b illustrates several features of this reverse engineering approach.

First, the

insoluble melanin-containing sample (e.g., melanin ghosts) is localized adjacent to the electrode by entrapping it in a thin, permeable and non-conducting hydrogel film of the aminopolysaccharide chitosan. Second, soluble mediators are purposely added to permeate throughout the film and shuttle electrons between the melanin sample and the underlying electrode. Third, a complex sequence of voltage inputs is imposed to the electrode, to enable the mediators to undergo electrochemical oxidation/reduction reactions. These mediators diffuse from the electrode surface into the film, exchange electrons with the entrapped melanin, and then return to the electrode where they undergo additional electrochemical reduction/oxidation reactions. Fourth, the electron transfer reactions between the mediator and electrode result in electrochemical currents and analysis of these current response characteristics can be related to the redox activities of the entrapped melanin. As illustrated in Scheme 1b, it is possible to select mediators in which the transfer of an individual electron at the electrode results in the generation of a diffusible free radical and this radical sometimes can have a distinct optical signal (i.e., color).33 Scheme 1b indicates that by 5


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monitoring the generation and loss of this optical signal it is possible to detect and characterize melanin’s ability to quench this free radical. Specifically, Scheme 1b illustrates a redox-cycling reaction in which (a) mediator-electrode electron transfer generates the colored free radical that can diffuse into the film, and (b) mediator-melanin electron transfer quenches the free radical to form the colorless non-radical form of the mediator. Thus by coupling electrochemical and optical measurements, it is possible to characterize melanin’s redox activities and its abilities to scavenge free radicals by an exchange of the electrons (i.e., by donating or accepting an electron to quench a radical).19, 34 (a) Putative functions of fungal melanin Melanin

Protection against environmental insults (Radiation) Cell death Pathogen-host interaction (Virulence/Defense)

Melanin in cell wall reduces injury

Non-Melanized

(b)

Melanized

Spectro-electrochemical reverse engineering Electrochemical output (Current)

Light

Insoluble fungal melanin ghosts

Radical scavenging Redox-cycling Chitosan film

Input (Volts) Mediator+•

Mediator

Electrode

Optical output (Absorbance)

e-

Scheme 1. (a) Melanins are often proposed to perform protective functions. (b) Spectroelectrochemical reverse engineering allows the redox and radical scavenging properties of melanin to be probed with diffusible mediators and complex voltage inputs, 6


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while the response characteristics are observed by both electrochemical and optical modalities. Using this spectroelectrochemical reverse engineering method, we observed that fungal melanin ghosts: are redox-active and can rapidly and repeatedly exchange electrons with soluble mediators; can scavenge free radicals either by donating or accepting electrons; and have redox and radical scavenging activities that are linked such that the melanin must be in a reduced state to quench radicals by donating electrons, or must be in an oxidized state to quench radicals by accepting electrons.

MATERIALS AND METHODS

Chemicals.

The followings were purchased from Sigma-Aldrich: chitosan, Ru(NH3)6Cl3

(Ru3+), melanin from Sepia officinalis, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and paraquat (PQ2+). 1,1’-ferrocenedimethanol (Fc) was purchased from Acros organics. The water (>18 MΩ) used in this study was obtained from a Super Q water system (Millipore). Chitosan solutions (1%, pH 5.5) were prepared by dissolving chitosan flakes in HCl to achieve a final pH of 5-6. The solutions of mediator were prepared in phosphate buffer (0.1 M; pH 7.0). Microorganisms. Single colonies of the fungal strain Wangiella dermatitidis 8656 (ATCC 34100, CBS 525.76) from YPD (2% peptone, 1% Bacto Yeast extract and 2% dextrose) agar plates were inoculated into 20 mL YPD broth and cultured at 25 °C with shaking at 200 rpm for 48 hours. Cell cultures were transferred (1:100) in 2 L YPD and grown with shaking at 37 °C for one week. Black yeast cells were harvested by centrifugation at 5000 rpm for 10 min. Isolation 7


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and purification of fungal melanin ghosts follows the method described by Dadachova et al.32 The resulting melanin ghosts were suspended in deionized water. Preparation of Film-Coated Electrode. To prepare the melanin–chitosan film, Sepia melanin (5 mg/mL) or fungal DHN melanin ghosts (100 mg/mL) were suspended in the chitosan solution (0.5%, pH 5.5), and 20 µL of this suspension was spread onto the standard gold electrode (radius = 1 mm). For spectroelectrochemical experiment, 30 µL of this suspension was dropped on the honeycomb electrode (Pine research instrumentation, NC) and the electrode was turned over to dry. The films were vacuum-dried at room temperature for 15 min and then immersed in phosphate buffer (0.1 M, pH 7.0; 10 min) to neutralize the chitosan and enable it to form its gel. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy. Sepia melanin or fungal DHN melanin ghosts were suspended in water and then spread onto gold substrates after which the substrates were dried. For the elemental analysis, energy dispersive Xray (SEM-EDX) spectroscopy was performed using a Bruker SOL-XE detector (Bruker AXS Inc.). The elemental analysis was obtained by averaging the analyzed values at three different points of each sample. For SEM imaging, all samples were coated with Au under argon plasma using a Hummer X sputter coater (Anatech, Springfield, VA) and the images were obtained using a scanning electron microscope (SEM, SU-70, Hitachi, Pleasanton, CA). Transmission Electron Microscopy (TEM). Sepia melanin or DHN-fungal melanin ghosts suspended in water were transferred to 1% low melting point agarose. The cooled stabilized pellet was immersed in 0.1M sodium cacodylate buffer overnight. The cells were subjected to post-fixation in 1% osmium tetroxide in 0.1M sodium cacodylate for 60 min at room temperature. Cells were then washed with distilled water and dehydrated through a 15 min immersion in graded ethanol series from 30% to 100%, with the 100% ethanol being changed 3 8


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times. A solution of propylene oxide (PO) was then added to a 1:1 ratio with the 100% ethanol for 15 min, followed by 3 changes of 100% PO. Infiltration was completed in Epon araldite resin containing 3 successive reductions of PO (PO:Epon araldite ratio: 2:1, 1:1, 1:2) for 1 hr each, before being transferred to 100% PO:Epon araldite and stored overnight at room temperature. The sample was then transferred to fresh resin in the embedding mold and polymerized at 65°C for 24 hrs. Blocks were sectioned to 90 nm on a Leica Ultracut Ultramicrotome and sections were placed on copper 200 mesh grids. The samples were viewed on a Hitachi HT7700 transmission Electron Microscope at 80kV. Electron Paramagnetic Resonance (EPR). To measure the EPR spectra of melanin free radical, melanin was suspended in water (10 mg/mL), which was placed into a capillary EPR tube and then its EPR spectra was measured. To observe the photo-response of melanin, light above 350 nm was generated by a Xenon lamp coupled with a filter WG320. The EPR signal was recorded by fixing the magnetic field of the first derivative maxima of melanin free radical and switching the light illumination with an interval of 60 s.

Next, to check the radical

scavenging properties of melanin, electrochemically oxidized ABTS+• solution was prepared by immersing the gold electrode (area = 1 cm2) in 0.1 M phosphate buffer solution (pH 7.0) containing 0.1 mM ABTS and a constant potential and applying to +0.7 V vs Ag/AgCl for 30 min with stirring. The resulting ABTS+• solution (50 µL) was placed into a capillary EPR tube. EPR spectra of ABTS+• solutions were measured at 1 minute intervals for 10 minutes. EPR spectra of other samples were obtained by suspending lyophilized power derived from melanindeficient fungal mutant wdpks1 cells (0.1 mg/mL), Sepia melanin (0.1 mg/mL) or fungal DHN melanin ghosts (0.1 mg/mL) in ABTS+• solution. After mixing, the mixture was placed into a capillary tube and EPR spectra were measured at 1 minute intervals for 10 min. All EPR 9


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measurements were performed out using a Bruker EMX ESR spectrometer (Billerica, MA) at ambient temperature. Electrochemical Instruments. Electrochemical measurements (cyclic voltammetry) were performed using a three electrode system with Ag/AgCl as a reference electrode and Pt wire as a counter electrode (CHI Instruments 600C electrochemical analyzer). Air was excluded by purging N2 during the experiment. Spectroelectrochemical Instruments.

The honeycomb electrode contains two gold

electrodes. One gold electrode with holes was used as a working electrode and the other patterned gold electrode was used as the counter electrode. The working electrode in honeycomb electrode was prepared by coating chitosan or melanin-chitosan films on the electrode surface. The film-coated honeycomb electrode and Ag/AgCl reference electrode were placed into a spectroelectrochemical cell (i.e., a cuvette). N2 was purged during the experiment. All of electrodes were connected with an electrochemical analyzer. The optical absorbance was monitored over time at a fixed wavelength (before measurement, we waited 2 minute to allow the optical signal to stabilize). Simultaneously, the optical (absorbance) and electrochemical (current) output responses were individually recoded over time.

RESULTS Characterization of Fungal DHN Melanin and Sepia Melanin

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(a)

(b)

2 µm

2 µm

(c)

200 nm

(d)

2 µm

100 nm

100 nm

(e) Fungal ghosts (DHN-melanin)

Sepia melanin

Carbon (%)

80.2

71.6

Nitrogen (%)

9.3

11.2

Oxygen (%)

11.5

17.2

OH

Putative unit structure

OH

HO (COOH) HO

N H

Figure 1. Image and Chemical Analysis. (a) SEM and (b) TEM images of fungal ghosts indicate melanin is assembled as a granular structure on the outer (≈100 nm thick) cell wall region. (c) SEM and (d) TEM images of Sepia melanin indicate granular structure. (e) Chemical analysis35 of fungal ghosts1, 36 and Sepia melanin22, 37 using EDX.

In this study, we compared the fungal DHN melanin with the commonly-studied eumelanin from Sepia officinalis (cuttlefish). Fungal melanin ghosts were prepared by treating melanized fungus Wangiella dermatitidis with strong acid to dissolve away cellular components and leave an intact melanized cell wall. Previous studies29, 32 reported that this treatment of melanin does not affect its chemical and physical properties. The SEM image in Figure 1a shows these melanin ghosts are about 5 µm, while the TEM cross-section of Figure 1b indicates the melanin is assembled as a granular structure on the outer region of the cell wall (≈100 nm thick).32 For 11


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comparison, the SEM image of Figure 1c shows the Sepia melanin particle is approximately 5 µm and also has a granular structure (individual granules of 100-200 nm).25 The TEM image of Figure 1d shows the cross sectional image of an individual Sepia melanin granule. While the fungal and Sepia melanins share a granular structure, it is important to note that the fungal melanin ghosts possess a melanin shell surrounding an empty core while the Sepia melanin particles are filled. Chemical characterization of the fungal melanin ghosts and Sepia melanin was performed using Energy Dispersive X-ray spectroscopy (EDX). The table in Figure 1e shows the melanin ghosts contain less nitrogen (9.3 %) compared to Sepia melanin (11.2%). As illustrated by the putative structural units in Figure 1e, the DHN melanin is not expected to have nitrogen in its structure and presumably the observed nitrogen reflects that traces of cell wall components, such as chitin, still remain in the melanin ghosts. Some studies38-41 have indicated that chitin is a primary anchor for melanin deposition within the fungal cell wall, although additional diverse constituents may also be involved in the localization and maintenance of melanin within the complex cell wall.

Electrochemical Reverse Engineering of Melanin Characterization of redox properties of fungal DHN melanin ghosts Initial electrochemical reverse engineering studies illustrate the experimental approach used to detect fungal DHN melanin’s redox-activity. A melanin-containing film is prepared by first suspending melanin particles in a chitosan solution (0.5 %, pH 5.5) and then dropping a small amount of this suspension (20 µL) on the gold electrode. The film was vacuum-dried at room temperature for 15 min and then was immersed into a phosphate buffered (0.1 M, pH 7.0) 12


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solution containing two mediators (50 µM Fc, 50 µM Ru3+). Previous studies42-44 showed that such mild drying processes to prepare melanin samples do not affect the chemical composition of melanin. As illustrated by the upper schematic in Figure 2a, the Ru3+–mediator was selected because previous studies have shown it is capable of undergoing reductive redox-cycling reactions that shuttle electrons from the electrode to melanin.24, 25 The bottom schematic in Figure 2a illustrates that the Fc-mediator was selected because it has been previously observed to undergo oxidative redox-cycling reactions that serve to shuttle electrons from the melanin to the electrode.24,

25

The thermodynamic plot in Figure 2b illustrates that these electron transfer

reactions are thermodynamically constrained so electrons “flow” from more negative to more positive potentials. Importantly, this thermodynamic plot also shows the hypothesis that DHNmelanin is redox-active with a redox potential intermediate between the two mediators. The input potential curve in Figure 2b illustrates that these initial experiments were performed by imposing a voltage to the underlying electrode that oscillated between +0.5 V and -0.5 V (vs Ag/AgCl).

If, as hypothesized, the melanin sample is redox-active with a redox potential

intermediate between the two mediators, then this potential range should be capable of sequentially engaging the mediators in the two redox-cycling reactions. Specifically, when the imposed voltage becomes negative then Ru3+-reductive redox-cycling should occur, and when the voltage becomes positive Fc-oxidative redox-cycling should occur. The output current response for this experiment is shown in Figure 2b as a plot of i vs t (note Figure S1 of Supporting Information shows an alternative representation of the data as cyclic voltammograms, i vs E). There are three negative controls in Figure 2b: (i) a chitosan film (no melanin); (ii) chitosan film with lyophilized powder from melanin-deficient mutant wdpks1 cells of W. dermatitidis45, 46 (a control for non-melanin cellular components); and (iii) chitosan film 13


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with DHN fungal melanin ghosts probed without the Fc and Ru3+ mediators. Figure 2b shows small output peak currents for these negative controls.

(a)

Reductive redox-cycling Ru3+

Ru2+

e-

e-

E0 vs. Ag/AgCl – -0.6 e-0.4 Ru3+/Ru2+

Oxidative redox-cycling Fc

(b)

Fc+ e-

Melanin e-

e-

Input / Potential (V)

Output/ Current(A)

Ru3+

Fc/Ru3+,Chit Fc/Ru3+,Melanin-deficient cell-chit

1

0

0

0.4

Fc/Ru3+, Fungal DHN mel-chit PB only, Fungal DHN mel-chit

Fc

0.6

+

2

-0.2

0.2

Fc/Fc+

3

0

20

40

60

80 100 0

Time (s)

20

40 60 Time (s)

80

-1

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-2 -3

100

Figure 2. Electrochemical reverse engineering to detect redox-activity. (a) Schemes for reductive and oxidative redox-cycling reactions that allow mediators to exchange electrons with melanin. (b) Thermodynamic plot of electron “flow” and the experimentally imposed input potential (i.e., voltage) and observed output current response associated with Ru3+ and Fc mediators. Scan rate = 20 mV/s. The experimental film in Figure 2b is the fungal DHN melanin-chitosan film probed with the oscillating potential in the presence of both mediators. The output currents in Figure 2b show large peak currents for both Ru3+-reduction and Fc-oxidation. The high amplification of the reduction and oxidation currents is consistent with significant redox-cycling through the two reactions shown in Figure 2a. These results provide initial evidence that fungal DHN melanin is redox-active.

Reversibility of redox activity of fungal DHN melanin

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Next, we examined the stability of the fungal DHN melanin’s redox-activity by imposing oscillating potential inputs over a long time (45 cycles; 2 mV/s) as illustrated in Figure 3a. Figure 3b shows the current output for the chitosan film containing the fungal DHN melanin compared to the output of a control chitosan film. The fungal DHN melanin chitosan film shows considerable amplification of both Ru3+-reduction and Fc-oxidation currents. Further, these amplified output currents remain nearly steady over the 45 cycles of the experiment. Using the same experiment, Figure 3c shows Sepia melanin-containing films also have amplified Fc-oxidation and Ru3+-reduction currents (compared to control chitosan films). During the initial 20 cycles, the Ru3+-reduction currents were observed to progressively increase until reaching the final steady amplified value. Possibly, this slow approach to steady output may reflect two phenomena: diffusional limitations associated with the mediators penetrating into the core of the Sepia melanin; and binding of the Ru3+-mediator to the melanin resulting in an initial depletion of the Ru3+-mediator from the film.47 After these initial 20 cycles, the films containing Sepia melanin showed that the amplified currents remain steady over time (note Figure S2 of Supporting Information shows an alternative representation of the data as cyclic voltammograms, i vs E).

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(a)

0.8

E(V) vs Ag/AgCl

0.4

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Ru3+

0 -0.4 Fc

-0.8 0

2

4 6 8 Time (hr)

10

12

10

12

10

12

Fungal DHN melanin chit

Current (µA)

(b)

1.2

Fungal DHN melanin-chit

0.6 0 -0.6 Chit

-1.2 0

2

4 6 8 Time (hr)

Sepia melanin chit

(c) Current (µA)

Output / Current (A)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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1.2

Sepia melanin chit

0.6 0 -0.6 Chit

-1.2 0

2

4 6 8 Time (hr)

Figure 3. Evidence that fungal DHN melanin can be repeatedly oxidized and reduced. (a) Long term oscillating potential inputs allows the mediators to repeatedly probe the redox properties of film-entrapped melanin (scan rate 2mV/s). (b) Films containing fungal DHN melanin ghosts show steady amplifications of both Fc-oxidation and Ru3+-reduction currents over 45 cycles. (c) Films containing Sepia melanin show a gradual approach to steady output currents.

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In summary, the results in Figure 3 indicate that both the fungal DHN melanin and Sepia melanin are redox-active and can be repeatedly engaged in oxidative and reductive redox-cycling reactions that yield amplified output currents. In contrast to conventional methods29, 48, 49 where oxidants or reductants are used to demonstrate melanin’s redox activity, this electrochemical approach allows both oxidation and reduction reactions to be studied in the same experiment and also the reversibility of these redox reactions to be investigated.

The observed response

characteristics indicate that these melanins can be reversibly switched between oxidized and reduced redox states without observed losses over time. It is important to note that Fc and Ru3+ are weak oxidants and reductants with redox potentials within the physiological range.

If

stronger oxidants or reductants were used, it is possible that the melanin structure could be damaged and the reversible redox activities could be lost.

Dynamics of electron transfer reactions Figure 1 shows that the Sepia melanin and DHN fungal melanin ghosts have dramatically different nano- and micro-structures which should affect the accessibility of redox-active sites for mediator redoxcycling. Figure 4a illustrates that these differences can be probed by dynamic analysis with varying scan rates.25, 50 Experimentally, we probed films with both Fc and Ru3+ mediators and varied the scan rate of the imposed oscillating potential input (-0.5 V to +0.5 V; 2 to 1000 mV/s). For data analysis, Figure 4b shows we calculated the charge transferred during the Fc-oxidation and Ru3+-reduction portions of the CVs by integrating the observed currents (Q=∫i⋅dt). For all cases, Figure 4c shows that the mediatorbased charge transferred at the electrode decreases with increasing scan rate. As illustrated in Figure 4a, this progressive decrease in Q with scan rate occurs because the mediators have less time to diffuse through the film to exchange electrons with the electrode.

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Figure 4b also shows that Q values for the melanin-containing films were ratioed against the charge transferred for the control chitosan film to calculate amplification ratios (AR). Figure 4d shows AR values calculated for chitosan films containing Sepia melanin and fungal DHN melanin, and also includes previous results with a chitosan film with grafted catechol moieties.25 This catechol-chitosan film serves as a control in which the redox-active moieties are readily accessible to the mediators. The AR values for this catechol-chitosan control show peaks near 100 mV/s. The observation of a peak in AR is explained by considering the relative rates of mediator diffusion and reaction in the film, and the finite redoxcapacity of the catechol-chitosan films to donate or accept electrons.50 Specifically, the denominator in

the AR is the charge transferred for control chitosan film (QChit) which reflects the diffusion of mediators to the electrode. The numerator in the AR is the charge transferred for the melanincontaining film (QMelanin-chit) and has contributions from both mediator diffusion and redox cycling. In the regime of very low scan rates, AR can become small because there is sufficient time for the mediators to diffuse long distances (e.g., from the bulk solution) which increases the denominator term while the relative contribution of redox-cycling can become less important because of the finite capacity of the catechol-chitosan film to donate or accept electrons. As the scan rate is increased, and redox-cycling becomes comparatively more important (vs mediator diffusion) the AR becomes larger. In the regime of very high scan rates, there is limited time for both mediator diffusion of and redox-cycling and this can suppress AR. In contrast to the catechol-chitosan control with readily accessible redox-active moieties, the microstructure of the Sepia melanin suggests many of its redox-active sites will be within the particles and thus less accessible to the mediators as illustrated in Figure 4a. For this case, longer times (or slower scan rates) should be required for the mediators to access these internal sites and thus the AR peaks would be expected to shift to lower scan rates. Consistent with this expectation, Figure 4d shows the ARs are shifted to the left for films with the Sepia melanin 18


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(compared to the catechol-chitosan control) and a peak in AR is not apparent but could appear at a scan rate less than 1 mV/s. The images in Figure 1 suggest that the open microstructure of the fungal DHN melanin should yield intermediate properties, which means that the redox sites should be more accessible than for the Sepia melanin but less accessible than for the catechol-chitosan films. Consistent with this expectation, Figure 4d shows the response characteristics of the fungal DHN melanin is intermediate with peaks in AR values appearing near 10 mV/s. In summary, the response characteristics of the melanin-containing films to probing at varying scan rates is consistent with expectations from their observed microstructures and suggests that redox-active sites are distributed throughout the melanin particles.

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Melanin (Sample)

(a) Chitosan

Mediator penetration

film Electrode

Low frequency Potential input

High frequency Potential input Fungal DHN melanin

Sepia melanin

3

(b)

Melanin-Chit

QRED, Ru

Current (µA)

2

ARRED =

3

Melanin −Chit QRED , Ru

2

Chit QRED , Ru

Melanin-Chit

1

1

QRED, Fc

0

0 Melanin-Chit

QOX, Ru

-1 -2

AROX =

Melanin-Chit

QOX, Fc

-3

Sepia melanin-chit Catechol-chit Fungal DHN melanin-chit

5 4 3 2

20

Chit

1 0

60

80

100

8

Sepia melanin-chit Catechol-chit Fungal DHN melanin-chit

6 4 2

Chit

0 1

6

10 100 1000 Scan rate (mV/s)

1 6

Sepia melanin-chit Catechol-chit

4

ARRED

(d)

40

Time (s)

Cathodic charge (QRED, Ru / mC/cm2)

Anodic charge (QOX, Fc / mC/cm2)

6

-2

Chit QOX , Fc

-3

00 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 E(V) vs Ag/AgCl

(c)

-1

Melanin −Chit QOX , Fc

Current (µA)

Mediator

AROX

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Fungal DHN-melanin chit

2

Sepia melanin-chit Catechol-chit

4

Fungal DHN-Melanin Chit

2

0

10 100 1000 Scan rate (mV/s)

0 1

10 100 1000 Scan rate (mV/s)

1

10 100 1000 Scan rate (mV/s)

Figure 4. Differences in dynamic responses due to differences in melanin’s micro/nano-structure. (a) Schematic illustrating that scan rate affects the depth of mediator penetration into the film and into the core of melanin particles. (b) Analysis of output curves to quantify charge transferred (Q) and amplification ratios (ARs). (c) 20


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Charge transferred and (d) amplification ratios (ARs) for Fc-oxidation and Ru3+-reduction for various films probed at different scan rates. Note: results for the catechol-chitosan control were reproduced with permission from reference.25 Copyright (2014) American Chemical Society Melanin as a Stable Radical One characteristic feature of natural melanin is the presence of stable free radicals, which can be detected by its distinctive signals in electron paramagnetic resonance (EPR) spectroscopy25, 51, 52

. Figure 5a shows the EPR spectra for chitosan solutions (0.5%) mixed with various powders

including Sepia melanin (10 mg/mL), fungal DHN melanin (10 mg/mL) or lyophilized powder derived from melanin-deficient cells (10 mg/mL), compared to a chitosan solution (0.5%). Chitosan solutions with either Sepia or fungal DHN melanin show a single EPR peak with a gfactor of 2.00369 (Sepia melanin) and 2.00348 (fungal DHN melanin), which confirms the presence of a stable free radical in melanin. For controls, the chitosan and melanin-deficient lyophilized fungal cells, showed no such EPR peak. A second characteristic feature of natural melanin is that free-radicals are generated in response to UV illumination.53, 54 To observe such a response, we fixed the magnetic field to the first-derivative maxima of the melanin radical, and intermittently illuminated the sample solutions with light of wavelengths above 350 nm. Figure 5b shows that the Sepia melanin and fungal DHN melanin-mixed chitosan solutions respond to this illumination. When light is on, the EPR peak immediately increases, which is consistent with free-radical generation. After 60 s, the illumination was turned off, and the EPR signal was observed to decrease, consistent with a decay of free radicals. This procedure was repeated, yielding similar results. Thus, the results in

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Figure 5 indicate that both Sepia melanin and fungal DHN melanin exhibit the characteristic free-radical features of melanin. (a) Signal

Sepia melanin-chit Fungal DHN Melanin-Chit Melanin-deficient cells -chit Chit

3355

(b)

3365 3375 3385 Magnetic Field (G) ON

Light ON

Signal

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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OFF

OFF Sepia melanin-chit Fungal DHN melanin-chit Melanin-deficient cells-chit Chit

0

60

120

180

240

Time (s)

Figure 5. The presence of stable free radical in melanin. (a) EPR spectra for Sepia melanin, fungal DHN melanin, or melanin-deficient lyophilized fungal cells mixed with chitosan solutions compared to control chitosan solution. (b) Free radical generation of melanin in response to UV illumination. Radical Scavenging Activity of Melanin EPR study 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) is commonly used to measure free

radical scavenging activities in melanin,55 food and agricultural systems.56, 57 In conventional antioxidant assays, ABTS is first converted to its radical cation form (ABTS+•) by a chemical oxidant (e.g., NaS2O8) and the ability of an antioxidant to quench this radical is characterized by

22


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monitoring optical absorbance. Importantly, the ABTS+• radical is green-colored while the nonradical ABTS is colorless. Figure 6a illustrates that we adapted this method by using electrochemical oxidation to convert the colorless ABTS to the green-colored ABTS+•.58

Initial confirmation that

electrochemical oxidation can generate the ABTS+• radical is provided by the Electron Paramagnetic Resonance (EPR) Spectroscopy.59 The EPR spectra in Figure 6a shows electrochemical oxidation generates the green-colored ABTS+• radical, while the addition of melanin attenuates both the color and EPR signal consistent with expectations that melanin has radical-scavenging activities. We performed a more systematic set of radical scavenging experiments as shown in Figure 6b. To obtain ABTS+• solution, buffer solution containing 0.1 mM ABTS was added into an electrochemical cell and the anodic potential (+0.7 V vs Ag/AgCl) was applied to the gold working electrode. After 30 min of reaction in this mixed electrochemical cell, the colorless solution was observed to become dark green, and Figure 6b shows a strong EPR peak emerges with g-factor of 2.00359. These results confirm that the electrochemical oxidation can generate the stable ABTS+• free radical. To demonstrate radical scavenging activities, we electrochemically generated the ABTS+• radical cation (as described above), incubated these solutions with samples for 5 min, and then measured EPR spectra. When melanin was tested, we used small amounts (0.1 mg/mL) compared to the amounts used in Figure 5 (10 mg/mL) to ensure that the observed EPR peak was primarily due to ABTS+• radical cation (EPR spectra for 0.1mg/mL melanin are shown in Figure S3 of Supporting Information). Figure 6b shows results for two controls in which either a chitosan solution (0.5 %) or lyophilized powder derived from melanin-deficient fungal cells (0.1 23


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mg) were mixed with the ABTS+• solution. Figure 6b shows little attenuation of the EPR peaks for these two controls which indicates that neither chitosan nor melanin-deficient lyophilized fungal cells possess significant ABTS+• radical cation quenching activity. When Sepia melanin (0.1 mg) was tested, Figure 6b shows the EPR peak for ABTS+• was attenuated by 43% which is consistent with its previously-reported radical scavenging activity.53,

60

When fungal DHN

melanin ghosts (0.1 mg) were tested, a 53% attenuation of the EPR peak for ABTS+• was observed. Finally, Figure 6c shows similar results when EPR peaks for ABTS+• were measured over time. Thus, traditional EPR methods for detecting free radicals demonstrate that both Sepia melanin and fungal DHN melanin can quench the ABTS+• free radical.

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(a)

Electrochemical generation of ABTS+• (radical) ABTS

e-

Quenching of ABTS+•

ABTS+•

Melanin

EPR measurement

ABTS+• ABTS+• /Chit ABTS+• /Melanin-deficient cells

(b) Signal

ABTS (0.5 mM) ABTS+• / Fungal DHN melanin ABTS+•/Sepia melanin

3335

3355

3375

3395

Magnetic Field (G)

(c) Relative Signal

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.2 1

ABTS+• ABTS+• /Chit ABTS+• / Melanin-deficient cells

0.8 0.6 0.4

ABTS+•/Sepia melanin ABTS+• /Fungal DHN Melanin

0.2 0 0

2

4 6 8 Time (min)

10

Figure 6. Melanins possess ABTS radical scavenging activities. (a) Schematic illustrating that electrochemical oxidation generates the green-colored ABTS+• radical while melanins can quench these radicals. (b) EPR spectra of the ABTS+• radical 5 min after mixing with various components: both fungal DHN melanin ghosts and Sepia melanin samples scavenge the ABTS+• radical. (c) Decay of EPR signal for solutions containing Sepia melanin or fungal ghosts with DHN-melanin, and control solutions containing chitosan or lyophilized fungal cells that lack melanin (designated melanindeficient cells). 25


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Page 26 of 50

Spectroelectrochemical Reverse Engineering to Probe Radical Scavenging Activity of Melanin Scavenging of oxidizing radical (ABTS+•) We next coupled the electrochemical measurements of redox-activities with the optical measurement of ABTS+• radical-scavenging activities using the honeycomb electrode of Figure 7a.61 The small holes in this gold electrode allow light to pass through for optical measurements in the vicinity of the electrode surface. Experimentally, chitosan films (with or without melanin) were coated on the honeycomb electrode and then these coated electrodes were immersed into a phosphate buffer solution containing 50 µM ABTS and 50 µM Ru3+. The thermodynamic plot in Figure 7a shows that under reducing voltages, Ru3+ can undergo reductive redox-cycling reactions to transfer electrons to melanin.

Under oxidative voltages, ABTS can be

electrochemically oxidized to ABTS+• and if this radical can be quenched by accepting electrons from melanin, then this should result in oxidative redox-cycling reactions as indicated in Figure 7a. To test for these redox-cycling reactions, the upper plot in Figure 7b shows that we imposed a voltage input to the underlying electrode to oscillate between +0.7 V and -0.4 V (vs Ag/AgCl). The middle plot of Figure 7b shows the output electrochemical (i.e., current) responses for electrodes coated with films of chitosan, Sepia melanin-chitosan, or fungal DHN melanin chitosan. The control chitosan film shows small peak currents for ABTS-oxidation and Ru3+reduction. When the chitosan film contained Sepia melanin (5 mg/mL) or fungal DHN melanin ghosts (100 mg/mL) the oxidation and reduction currents were amplified consistent with redoxcycling reactions.

26


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The lower plot in Figure 7b shows the optical output responses that were measured simultaneously with the electrochemical outputs.

Results for the control chitosan-coated

electrode show that when oxidizing voltages were imposed, a considerable optical absorbance was observed to emerge at 420 nm (Figure S4 of Supporting Information shows the UV-Vis spectrum for the anodically-generated ABTS+•). This increase in absorbance for the control chitosan film indicates that the green colored ABTS+• is formed at the electrode and diffuses into the optical path. When the chitosan film contained either Sepia-melanin or fungal DHN melanin ghosts, a much lower optical output responses were observed. An attenuation of the optical signal is expected if the electrochemically-generated ABTS+• is rapidly quenched by the melanin.

These results are consistent with the EPR results of Figure 6 and indicate that

spectroelectrochemistry provides a simple approach to dynamically monitor melanin’s radicalscavenging activities.

Correlation between redox-activity and radical-scavenging activity The amplified electrical signal and attenuated optical signal are consistent with an ABTS oxidative redox-cycling mechanism that links melanin’s redox and radical scavenging activities. To further examine the correlation between redox and radical scavenging activities, we probed chitosan films containing various amounts of fungal DHN melanin ghosts in the presence of 50 µM ABTS and 50 µM Ru3+. Figure 7c shows the oscillating input voltage (upper plot) while the electrochemical output responses (middle plot) show that both the ABTS-oxidation and Ru3+reduction currents increased with increasing levels of fungal DHN melanin ghosts. The optical output responses in the lower plot in Figure 7c show increasing attenuation of ABTS+• absorbance with increasing levels of fungal DHN melanin ghosts. 27


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Page 28 of 50

To demonstrate a correlation between melanin’s redox and radical scavenging activities, we prepared the cross-plot of Figure 7d between the independent electrochemical and optical measurements. To estimate the number of electrons transferred to the fungal DHN melanin (Ne), we calculated the difference in oxidative electron transfer between the melanin-containing and the control chitosan film (this difference is associated with ABTS+• oxidative redox-cycling: details of this calculation are shown in Figure S5 of Supporting Information). The radicalscavenging activity was quantified as the fractional attenuation of the optical absorbance between the melanin-containing and control chitosan films (details of this calculation are shown in Figure S6 of Supporting Information). Figure 7d shows a linear relationship between radicalscavenging and redox-activities and indicates that melanin’s ability to donate electrons to quench the ABTS+• radical is correlated to its oxidative redox-activity.

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Electrode for spectro-electrochemistry

E0 vs. Ag/AgCl –

e-

– E0Melanin

Melanin

Melanin

ABTS/ABTS+••

ABTS+•

Generation of oxidizing radical

+ Ru3+ + e- Ru2+

-0.4 -0.2 0

Output / Optical

E(V) vs Ag/AgCl

ABTS ABTS+• + e-

0.2 0.4

Current (µA)

0.6 20 0.8

-0.2 0 0.2 0.4

10

30 15 0

100 mg-DHN-melanin 50 mg-DHN-melanin 25 mg-DHN-melanin 10 mg-DHN-melanin

Amplify electrochemical signal

Chit

Redox activity

-15

Reducing of melanin-film

-30 0.12 Chit

0.1

0

Sepia melanin chit

Amplify electrochemical Signal


0.08 0.06

25 mg-DHN-melanin

0.04

50 mg-DHN-melanin

0.02

100 mg-DHN-melanin

0

(d)

0.2 Chit

0.15

Quenching of oxidizing radical

Attenuate optical Signal

0.1 0.05

Radical scavenging activity

0


0.25 -20

Attenuate optical signal

10 mg-DHN-melanin

1

Chit

-10

Absorbance at 420 nm

Output / Electrochemical


-0.6

-0.4

0.8

– +0.5 V

e-

(b)

Ru3+ + e- Ru2+

0.6

e-

Current (µA)

ABTS

Quenching of oxidizing radical


Light beam path


e-

Ru2+/3+

(c) – -0. 2 V

E(V) vs Ag/AgCl

Reducing Melanin

ABTS ABTS+• + e-

-0.6


e-

2 3 Time (min)

1.2

Relative Radical scavenging activity

(a)

Output / Optical

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

y = 0.0417x R² = 0.9869

1 0.8 0.6 0.4 0.2 0 0

Sepia melanin chit Fungal DHN Melanin Chit

10

20

30

Redox activity (Ne(nmol/cm2))

0 0

5

10

15

Time (min)

Figure 7. Spectroelectrochemical reverse engineering to detect quenching of oxidative radical. (a) Schematic illustrating that a honeycomb electrode is coated with a melanin-containing film and this film is probed using one mediator (Ru3+) to reduce melanin and a second mediator (ABTS) that is electrochemically-oxidized to generate the ABTS+• radical. (b) Potential input and output responses show melanin-containing films amplify the electrochemical currents and attenuate the optical absorbance of the ABTS+• 29


Biomacromolecules

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radical (scan rate = 2 mV/s). (c) Potential input, and electrochemical and optical output responses for films containing various amounts of fungal DHN melanin ghosts (scan rate =10 mV/s). (d) Correlation between redox activity and the ABTS+• radical scavenging activity. Reversibility of oxidizing radical scavenging activity To more rigorously examine the correlation between redox and radical scavenging activities, we performed longer-term, multi-cycle spectroelectrochemical experiments with fungal DHN melanin using the input voltage sequence shown in Figure 8a. The plots at the left in Figure 8 represent the case of near steady response characteristics. In this steady case, the imposed potential range (+0.7 V ∼ -0.4 V) is oscillated to enable both the oxidative redox-cycling of ABTS and the reductive redox-cycling of Ru3+. The electrochemical outputs in Figure 8b show two important response characteristics for this steady case: (i) both Ru3+-reduction currents and ABTS-oxidation currents are amplified; and (ii) these amplified current peaks remain nearly constant over the multiple cycles. These results are consistent with a conclusion that the fungal DHN melanin can be repeatedly oxidized and reduced by ABTS and Ru3+ redox-cycling. The optical outputs are shown in Figure 8c. For the control chitosan film, the absorbance associated with the ABTS+• radical is observed to increase when the voltage is cycled into the oxidative range (the ABTS+• radical is electrochemically generated) and then decreases when the voltage is cycled into the reduction range (the ABTS+• radical is electrochemically reduced). For the film containing fungal DHN melanin, the optical absorbance is greatly attenuated consistent with the conclusion that the electrochemically-generated ABTS+• radical is quenched by accepting an electron from fungal DHN melanin. 30


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The plots at the right in Figure 8 represent the case of unsteady response characteristics. In this unsteady case, Figure 8a shows a more limited potential range was imposed (+0.7 V ∼ 0 V) to provide the oxidative voltages required to oxidize ABTS and enable its redox-cycling, but to provide reducing voltages that are insufficient to reduce Ru3+ and enable its reductive redoxcycling. Under these unsteady conditions, electrons should be progressively depleted from the entrapped melanin. As expected, Figure 8b shows the electrochemical output response for this unsteady case has no Ru3+ reduction peaks for either the control chitosan or the fungal DHN melanin -containing films. Further, Figure 8b shows that initially the ABTS oxidation peaks are observed to be amplified with the fungal DHN melanin. However, these ABTS oxidation peaks are observed to progressively decrease over time approaching the peaks observed for the control chitosan film. Presumably, this decrease in ABTS oxidation currents is due to the progressive depletion of electrons from the melanin which prevents it from donating electrons to ABTS+• for continued reductive redox-cycling. The optical outputs in Figure 8c show that for the unsteady case, the absorbance of the ABTS+• radical is initially very small with the fungal DHN melanin sample which is consistent with a rapid quenching by the melanin. Over time, the ABTS+• absorbance is observed to increase approaching the signal observed for the control chitosan film (i.e., the optical signal becomes less attenuated). This unsteady optical response indicates that the electrochemically-generated ABTS+• radical is initially quenched by accepting electrons from melanin, but over time as the melanin becomes progressively depleted of electrons, it is no longer able to donate electrons to quench the ABTS+• radical. The results from this unsteady case indicate that as the fungal DHN melanin is progressively depleted of electrons it loses its ability to donate electrons for ABTS+• radical scavenging. 31


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8d shows a correlation between the radical scavenging and redox activities analogous to that shown in Figure 7d. Specifically, the radical scavenging activity is measured by the optical attenuation while the redox activity is measured by the difference in ABTS oxidative charge transferred between the film containing fungal DHN melanin and the control chitosan film. The correlation in Figure 8d indicates that melanin’s ability to quench the ABTS+• radical requires that it has donatable electrons.

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Page 33 of 50

Steady

Unsteady

(a)

Input / Potential (V) -0.6

Ru3+

-0.3

Current (µA)

E(V) vs Ag/AgCl

-0.6

0 0.3

-0.3

ABTS

0

0

90

15 30 Time (min)

45

Output / Electrochemical 30

30 Ru3+

Current (µA)

10 0 -10 -20

Fungal DHN melanin-chit

20

20

Current (µA)

0.3

0.9

30 60 Time (min)

(b)

10 0 -10 -20

ABTS

Chit

-30

-30 0

30 60 Time (min)

(c)

0

90

15 30 Time (min)

45

Output / Optical Absorbance at 420 nm

0.1 0.08 0.06 0.04 0.02 0

0.1

Fungal DHN melanin-chit Chit

0.08 0.06 0.04 0.02 0

30 60 Time (min)


0

(d)

0

0.6

0.6 0.9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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90

0

15 30 Time (min)

45

1 0.8

Steady (Fungal DHN melanin chit)

0.6 0.4 0.2

Unsteady (Fungal DHN melanin chit)

0 0 5 10 15 Redox activity (Ne(nmol/cm2))

Figure 8. Evidence for repeated quenching of oxidative radical (a) Long term oscillating potential input used to probe for ABTS+• radical scavenging by chitosan films containing fungal DHN melanin ghosts (scan rate= 10 mV/s). (b) Electrochemical and (c) 33


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optical outputs in the presence of both Ru3+ and ABTS using imposed potential range that either allows (left) or precludes (right) Ru3+ reductive redox-cycling. (d) Correlation linking redox and radical scavenging activities. Scavenging of reducing radical (PQ+•) Figure 8 illustrates melanin’s ability to quench radicals by donating electrons and we next examined melanin’s ability to quench radicals by accepting electrons. Figure 9a shows that paraquat (PQ2+) can be electrochemically reduced to generate the PQ+• free radical.

The

thermodynamic plot in Figure 9a suggests a reductive-redox cycling mechanism in which electrons transferred from the electrode to PQ2+ can be subsequently transferred from the PQ+• radical to melanin. The transfer of electrons to melanin by reductive redox-cycling can reduce the melanin and to test this mechanism we added the oxidative redox-cycling mediator Fc to deplete electrons from the melanin under oxidative voltages. Importantly, this electron transfer radical-scavenging reaction can be monitored optically because the non-radical PQ2+ form is colorless while the reduced PQ+• radical has a blue color and strong absorbance at 394 nm (Figure S4 of Supporting Information shows the UV-Vis spectrum for the electrochemicallygenerated PQ+•). To experimentally test these redox-cycling reactions, the upper plot of Figure 9b shows that we imposed an oscillating input voltage between +0.5 V and -0.8 V in a solution containing both Fc and PQ2+ (100 µM each). The electrochemical output (i.e., current) responses of Figure 9b show the control chitosan film has small peak currents for Fc-oxidation and PQ2+-reduction while films containing Sepia melanin or fungal DHN melanin show amplified Fc-oxidation and PQ2+-reduction currents.

Compared to the results with ABTS oxidation in Figure 7, the 34


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measurement of PQ2+ reduction currents is less accurate because under these reducing voltages a relatively high background current is observed due to water electrolysis. This background in the electrochemical output limits the opportunity to observe amplifications or to quantify results in this reducing region. The optical output (i.e., absorbance at 394 nm) responses are shown in the lower plot of Figure 9b. A large increase in PQ+• absorbance is observed for the control chitosan film when the underlying electrode is cycled through reducing voltages. Films containing either Sepia melanin or fungal DHN melanin show much lower PQ+• absorbance. In summary, the amplified electrical outputs and attenuated optical outputs are consistent with a PQ2+ reductive redox-cycling mechanism and provides initial evidence that melanin can quench radicals by accepting electrons. To more rigorously examine the reductive redox-cycling and radical-scavenging activities, we performed longer-term, multi-cycle spectroelectrochemical experiments as in Figure 8 (See Figure S7 of Supporting Information). Under steady conditions, the electrochemical output shows paired and steady amplifications of PQ2+-reduction and Fc-oxidation currents consistent with reversible redox-activity, and the optical output shows nearly complete attenuation consistent with PQ+•-radical quenching.

Under unsteady conditions, the electrochemical

amplification and optical attenuation were observed to progressively decrease over time. Figure 9c shows the ability of fungal DHN melanin to quench PQ+•-radicals is correlated to its ability to accept electrons.

Thus, analogous to the conclusions with the oxidative radical (ABTS+•),

Figure 9c indicates that melanin’s redox and radical scavenging activities are linked for the reductive PQ+•-radical.

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(a)

E0 vs. Ag/AgCl –

e-

e-

Generation of Reducing Radical

PQ2+

– -0.63 V

ePQ+•

PQ1+••/PQ2+

Quenching of Reducing Radical

Melanin

Melanin

e-

Oxidizing Melanin

– E0Melanin e– +0.25 V

Fc+/Fc

+

(b)

Fc Fc+ + e-


E(V) vs Ag/AgCl

-1

PQ2+ + e- PQ+•

-0.5 0 0.5

Current (µA)


10

Generation of reducing radical

Chit

0 Sepia melanin chit

-10

Oxidation of melanin-film



Output / Optical


20

-20

0.14 Attenuate optical signal

Chit

0.09

Quenching of reducing radical

Sepia melanin chit

0.04


-0.01

0

5

10

15

20

Time (min)

(c)

1.2


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Steady (Fungal DHN melanin chit)

1 0.8 0.6

Unsteady (Fungal DHN melanin chit)

0.4 0.2 0 0 10 20 Redox activity (Ne(nmol/cm2))

Figure 9. Spectroelectrochemical evidence for the quenching of reductive radical (a) Schematic illustrating that an electrode is coated with a melanin-containing film and is 36


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probed using one mediator (Fc) to oxidize melanin and a second mediator (PQ2+) that is electrochemically-reduced to generate the PQ+• radical. (b) Potential input and output responses show melanin-containing films amplify the electrochemical currents and attenuate the optical absorbance of the PQ+• free radical (scan rate = 2 mV/s). (c) Correlation linking redox and radical scavenging activities. Scavenging of reducing and oxidizing radicals Using independent redox-cycling systems, the above results demonstrate that melanin can quench radicals either by donating electrons to an oxidizing radical (i.e., ABTS+•) or accepting electrons from a reducing radical (i.e., PQ+•). In the final experiments, we investigated a single system in which both the oxidative and reductive redox-cycling reactions involve radical scavenging. The thermodynamic plot for this system is shown in Figure 10a. Experimentally, we used a mixed solution of PQ2+ and ABTS (50 µM each) and the upper plot of Figure 10b shows that we cycled the imposed voltage between +0.7 V and -0.8 V which is sufficient to induce ABTS redox-cycling under oxidative potentials and PQ2+ redox-cycling under reducing potentials. The electrochemical output is shown in the middle plot of Figure 10b. Results for the control chitosan film show small electrochemical currents for ABTS oxidation and PQ2+ reduction while peak currents for films containing either Sepia melanin or fungal DHN melanin show amplified redox currents consistent with oxidative and reductive redox-cycling. The optical output is shown in the lower plot of Figure 10b. Because the UV-VIS spectra of ABTS+• and PQ+• partially overlap, absorbance measurements at a single wavelength (394 nm) can observe the sequential emergence and disappearance of these two radicals (Figure S4 of Supporting 37


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Information shows UV-Vis spectra).

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For the control chitosan film, absorption peaks are

observed both during the oxidative portion of the cycle (due to generation of the ABTS+•radical) and during the reductive portion of the cycle (due to generation of the PQ+•-radical). For films containing either Sepia melanin or fungal DHN melanin, Figure 10b shows the optical output is attenuated both during the oxidative and the reductive portions of cycle. Attenuation of these two optical signals is consistent with the sequential quenching of the oxidative radical (ABTS+•) and the reductive radical (PQ+•).

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(a)

E0 vs. Ag/AgCl –

Reductive Redox-Cycling e-

eGeneration of Reducing Radical

PQ2+

e-

– -0. 63 V

1+• •/ 2+ Reducing Melanin PQ PQ

PQ+• Quenching of Melanin

ABTS

Reducing Radical

e-

Melanin Quenching of Oxidizing Radical Oxidizing Melanin

Melanin

– E0Melanin e– +0.5 V

ABTS/ABTS+••

ABTS+•

Generation of Oxidizing Radical

eOxidative Redox-Cycling

+

ABTS ABTS+• + e- PQ2+ + e- PQ+•

-1

E(V) vs Ag/AgCl


(b)

-0.5 0 0.5

Current (µA) Absorbance at 394 nm


20 1

Output / Optical

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Generation of reducing radical

10


0 Chit

-10

Sepia melanin chit



0.35 -20 0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05

Chit

Attenuate optical signal Sepia melanin chit

Scavenging of radical

Fungal DHN melanin Chit

0

5

10

15

20

25

Time (min)

Figure 10. Melanin can scavenge radicals by both donating and accepting electrons (a) Schematic illustrating that melanin can quench radicals by accepting electrons (e.g., from PQ+•) or donating electrons (e.g., to ABTS+•). (b) Potential input and output responses show melanin-containing films amplify the electrochemical currents and

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attenuate the optical absorbance for both the PQ+• radical and ABTS+• radical (scan rate = 2 mV/s). Longer-term, multi-cycle spectroelectrochemical experiments were performed under steady conditions to test the reversibility of these radical scavenging activities. Specifically, we probed the films using a mixed solution containing both PQ2+ and ABTS (50 µM each) and the imposed input voltages (+0.7 V ~ -0.8 V) shown in Figure 11. The output electrochemical responses show amplified and steady currents for both PQ2+ reduction and ABTS oxidation.

These

responses indicate that melanin can repeatedly accept electrons from the electrochemically generated PQ+•-radical and donate electrons to the electrochemically-generated ABTS+•-radical. The output optical responses in Figure 11 for the control chitosan film show large peak absorbances (measured at 394 nm) for both the PQ+• and ABTS+• radicals. These optical signals were essentially absent when the film containing the fungal DHN melanin was probed. This attenuation in the optical signals indicates that the fungal DHN melanin can sequentially quench both the ABTS+• oxidative radical and the PQ+• reductive radical.

Input / Potential (V) e– 2+-0.63 V

e-

PQ1+••/PQ Quenching of Reducing Radical Melanin eQuenching of Oxidizing Radical ABTS/ABTS+•• Generation of Oxidizing Radical

0 – E Melanin

e– +0.5 V

PQ2+

-1

30

Output / Optical

Chit

20

-0.5

Current (µA)

Generation of Reducing Radical



Steady

E(V) vs Ag/AgCl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 0.5

10 0 -10

ABTS


-20

1 0

30 60 Time (min)

90

0

30 60 Time (min)

90

Chit Fungal DHN melanin-chit

0.2

0.1

0 0

30 60 Time (min)

Figure 11. Reversibility of melanin’s redox and radical scavenging activities. Schematic and input/output curves (scan rate = 10 mV/s) show that melanin can 40


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repeatedly scavenge radicals by sequentially accepting electrons (e.g., from PQ+•) and donating electrons (e.g., to ABTS+•).

DISCUSSION There are two novel aspects of this work. First is the continued development of a new experimental methodology based on electrochemical reverse engineering. Conventional methods to study oxidation and reduction reactions in biology rely on the addition of reagents and typically study reactions in one direction. Electrochemical reverse engineering allows redox conditions to be changed sequentially so both oxidative reactions and reductive reactions can be studied in the same experiment. Further, redox inputs can be sequenced repeatedly to probe the reversibility of redox reactions. Here, we extended this reverse engineering methodology from the measurement of a single electrochemical modality to the simultaneous measurement of a second optical modality. This extension is important because (i) electrochemistry provides a simple reagentless means to generate controlled amounts of free radicals, and (ii) optical measurements can often detect these free radicals because the radical and non-radical forms of a chemical often have distinct optical signatures. Thus, spectroelectrochemical analysis provides a unique opportunity to study the repeatability of radical scavenging activities in both oxidative and reductive directions. Also it is important that the instrumentation is relatively simple and inexpensive, and the approach is versatile as various input electrical signals can be imposed to probe specific questions. The current challenges are selecting electron shuttling mediators and designing input sequences that yield meaningful and interpretable outputs. The second novel aspect of this work is that we applied this spectroelectrochemical reverse engineering approach to insoluble melanins and demonstrate that they can repeatedly donate and 41


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accept electrons to quench free radicals. We further showed that melanins’ redox and radical scavenging activities are linked: the melanin must be reduced (i.e., have donatable electrons) to quench an oxidative free radical; or the melanin must be oxidized and capable of accepting electrons to quench a reductive free radical. We observed these redox and radical scavenging activities both for melanins derived from tyrosinase-catalyzed reactions (i.e., Sepia melanin) and for the fungal DHN melanin which is derived from a different polyketide pathway and has a markedly different chemical structure. Melanins are possibly the least understood macromolecules in biology and we believe that clarifying melanins’ redox and radical scavenging activities may be essential to understanding their biological functions. Previously, we demonstrated that pheomelanin has a more oxidative redox potential (vs eumelanin)27 which may explain its characteristic pro-oxidant effects: it can accept electrons from a wider range of biological reductants (e.g., ascorbate and glutathione)54, 62 and donate electrons to O2 to generate reactive oxygen species.25 63 Here, we observed rapid and repeatable radical scavenging activities which are integral to melanins’ protective biological functions. An obvious question is whether the unique ability of melanin to exist as a stable free radical (Figure 5) is related to its redox and radical scavenging activities? We have observed that melanins have redox potentials in the middle of the physiological range which means their in vivo behavior will be sensitive to redox context. Interestingly, melanins often exist at interfaces that are expected to have complex redox contexts that could promote the transfer of electrons from a reducing-side to an oxidizing-side. For instance, the fungal DHN melanin is positioned at the cell wall (Figure 1) which may be integral to its protective and pathogenic activities: possibly reducing equivalents from the biotic side of this interface can provide electrons that can protect the cell from extracellular oxidative insults associated with a 42


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contaminated environment (e.g., Chernobyl reactor) or an aggressive host response.11, 13, 14, 18 In an analogous fashion, the melanin generated as part of the insect’s innate immune response to encapsulate invading pathogens may serve as a redox-active interface between the host hemolymph and the encapsulated pathogen.16 Additional observations have stimulated speculations that melanins may perform additional, broader biological functions that would be linked to its redox activities. Separate groups working with different biological systems have independently suggested that melanin may perform energy harvesting activities although mechanisms are (currently) unknown.19, 64-66 In addition, observation that melanins exchange electrons with a broad range of diffusible redox-active species suggests they may play an active role in biological redox signaling and redox biology.6769

Experimental tools that can clarify melanins’ redox activities may be essential to investigating

these putative functions.

CONCLUSONS We report a spectroelectrochemical reverse engineering methodology that allows the simultaneous measurement of redox and radical scavenging activities. Using this methodology, we observed that melanins (and especially fungal DHN melanin) are redox active and can repeatedly scavenge radicals by donating and accepting electrons. Further, we demonstrate that these redox and radical scavenging activities are linked.

We believe this experimental

methodology provides a unique opportunity to clarify the redox properties of melanins that underpin many of its proposed biological functions. Potentially, this methodology could also provide an experimental screening tool to assess the properties of mimetic melanin materials generated either by chemical synthesis or synthetic biology. More broadly, we believe this work 43


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illustrates that electrochemistry can contribute valuable experimental capabilities to the study of redox biology.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: . Cyclic voltammograms as an alternative output display, UV-VIS spectra of electrochemically generated ABTS+• and PQ+•, EPR spectra of ABTS+• and melanins, calculation of melanin’s redox and radical scavenging activities, additional results of longer-term, spectroelectrochemical experiments to examine

the reductive redox-cycling and radical-

scavenging activities. (PDF)

AUTHOR INFORMATION Corresponding Author Gregory F. Payne e-mail: [email protected] Phone: 301-405-8389 FAX:

multi-cycle

301-314-9075

ORCID 44


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Gregory F. Payne: 0000-0001-6638-9459 Eunkyoung Kim: 0000-0003-2566-4041

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work has been supported by NSF (DMREF-1435957) and DTRA (HDTRA1-13-1- 0037, HDTRA1-15-1-0058). We acknowledge the support of the Maryland NanoCenter and its AIMLab. This paper is not an official U.S. FDA guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred.

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Biomacromolecules

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Biomacromolecules

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Table of Contents Graphic

Input (Electrical)

Radical Scavenging Redox-Cycling

e-

Mediator

Output (Optical)

Melanin

Output (Electrical) Input (Optical)

e-

Free Radical Mediator

eElectrode

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Spectroelectrochemical Reverse Engineering DemonstratesThat

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