Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO- occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB⋅), much longer-lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

Ultrafast photoreduction dynamics of a new class of CPD photolyases.

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100 ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950 ps and ~ 50 ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome.

The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y373 plays a crucial role by elongating , as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad. Here, we report the full mechanism of light-induced FADH• formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FAD•- by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WH•+ radical along the tryptophan triad (∼4 and ∼50 ps). Oxidation of Y373 by coupled ET to WH•+ and deprotonation then proceeds in ∼800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FAD•-/Y373• pair is formed with high quantum yield (∼60%); its intrinsic decay by recombination is slow (∼50 ms), favoring reduction of Y373• by extrinsic agents and protonation of FAD•- to form the long-lived, red-light absorbing FADH• species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 µs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Loss of Fourth Electron-Transferring Tryptophan in Animal (6-4) Photolyase Impairs DNA Repair Activity in Bacterial Cells.

(6-4) photolyases [(6-4)PLs] are flavoproteins that use blue light to repair the ultraviolet-induced pyrimidine(6-4)pyrimidone photoproduct in DNA. Their flavin adenine dinucleotide (FAD) cofactor can be reduced to its repair-active FADH- form by a photoinduced electron transfer reaction. In animal (6-4)PLs, a chain of four Trp residues was suggested to be involved in a stepwise transfer of an oxidation hole from the flavin to the surface of the protein. Here, we investigated the effect of mutation of the fourth Trp on the DNA photorepair activity of Xenopus laevis (6-4)PL (Xl64) in bacterial cells. The photoreduction and photorepair properties of this mutant protein were independently characterized in vitro. Our results demonstrate that the mutation of the fourth Trp in Xl64 drastically impairs the DNA repair activity in cells and that this effect is due to the inhibition of the photoreduction process. We thereby show that the photoreductive formation of FADH- through the Trp tetrad is essential for the biological function of the animal (6-4)PL. The role of the Trp cascade, and of the fourth Trp in particular, is discussed.

Ultrafast flavin photoreduction in an oxidized animal (6-4) photolyase through an unconventional tryptophan tetrad.

Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (∼0.5 ps) by a nearby tryptophan residue, yielding FAD˙- and WH˙+ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (∼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.

Energetics of Photoinduced Charge Migration within the Tryptophan Tetrad of an Animal (6-4) Photolyase.

Cryptochromes and photolyases are flavoproteins that undergo cascades of electron/hole transfers after excitation of the flavin cofactor. It was recently discovered that animal (6-4) photolyases, as well as animal cryptochromes, feature a chain of four tryptophan residues, while other members of the family contain merely a tryptophan triad. Transient absorption spectroscopy measurements on Xenopus laevis (6-4) photolyase have shown that the fourth residue is effectively involved in photoreduction but at the same time could not unequivocally ascertain the final redox state of this residue. In this article, polarizable molecular dynamics simulations and constrained density functional theory calculations are carried out to reveal the energetics of charge migration along the tryptophan tetrad. Migration toward the fourth tryptophan is found to be thermodynamically favorable. Electron transfer mechanisms are sought either through an incoherent hopping mechanism or through a multiple sites tunneling process. The Jortner-Bixon formulation of electron transfer (ET) theory is employed to characterize the hopping mechanism. The interplay between electron transfer and relaxation of protein and solvent is analyzed in detail. Our simulations confirm that ET in (6-4) photolyase proceeds out of equilibrium. Multiple site tunneling is modeled with the recently proposed flickering resonance mechanism. Given the position of energy levels and the distribution of electronic coupling values, tunneling over three tryptophan residues may become competitive in some cases, although a hopping mechanism is likely to be the dominant channel. For both reactive channels, computed rates are very sensitive to the starting protein configuration, suggesting that both can take place and eventually be mixed, depending on the state of the system when photoexcitation takes place.

Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy.

DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH(-) (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD(.-) and FADH(.) formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD(.-) (formed by ET from the tryptophan chain) in plant CRYs in about 1.5 µs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD(.-) is converted in 3.3 µs into a state with spectral features that are different from both FADH(.) and FAD(.-) . Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

ATP binding and aspartate protonation enhance photoinduced electron transfer in plant cryptochrome.

Cryptochromes are flavoproteins encountered in most vegetal and animal species. They play a role of blue-light receptors in plants and in invertebrates. The putative resting state of the FAD cofactor in these proteins is its fully oxidized form, FADox. Upon blue-light excitation, the isoalloxazine ring (ISO) may undergo an ultrafast reduction by a nearby tryptophan residue W400. This primary reduction triggers a cascade of electron and proton transfers, ultimately leading to the formation of the FADH° radical. A recent experimental study has shown that the yield of FADH° formation in Arabidopsis cryptochrome can be strongly modulated by ATP binding and by pH, affecting the protonation state of D396 (proton donor to FAD°(-)). Here we provide a detailed molecular analysis of these effects by means of combined classical molecular dynamics simulations and time-dependent density functional theory calculations. When ATP is present and D396 protonated, FAD remains in close contact with W400, thereby enhancing electron transfer (ET) from W400 to ISO*. In contrast, deprotonation of D396 and absence of ATP introduce flexibility to the photoactive site prior to FAD excitation, with the consequence of increased ISO-W400 distance and diminished tunneling rate by almost two orders of magnitude. We show that under these conditions, ET from the adenine moiety of FAD becomes a competitive relaxation pathway. Overall, our data suggest that the observed effects of ATP and pH on the FAD photoreduction find their roots in the earliest stage of the photoreduction process; i.e., ATP binding and the protonation state of D396 determine the preferred pathway of ISO* relaxation.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV.

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH(-)). The CPD repair mechanism involves electron transfer from photoexcited FADH(-) to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH(-). The two electron transfer processes occur on time scales of 10(-10) and 10(-9) s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T(∘-)) and electron return from T(∘-) to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Photo-oxidative damage of photosystem I by repetitive flashes and chilling stress in cucumber leaves.

Photosystem I (PSI) is an essential protein complex for oxygenic photosynthesis and is also known to be an important source of reactive oxygen species (ROS) in the light. When ROS are generated within PSI, the photosystem can be damaged. The so-called PSI photoinhibition is a lethal event for oxygenic phototrophs, and it is prevented by keeping the reaction center chlorophyll (P700) oxidized in excess light conditions. Whereas regulatory mechanisms for controlling P700 oxidation have been discovered already, the molecular mechanism of PSI photoinhibition is still unclear. Here, we characterized the damage mechanism of PSI photoinhibition by in vitro transient absorption and electron paramagnetic resonance (EPR) spectroscopy in isolated PSI from cucumber leaves. Photodamage to PSI was induced by two different light treatments: 1. continuous illumination with high light at low (chilling) temperature (C/LT) and 2. repetitive flashes at room temperature (F/RT). These samples were compared to samples that had been illuminated with high light at room temperature (C/RT). The [FeS] clusters FX and (FA FB) were destructed in C/LT but not in F/RT. Transient absorption spectroscopy indicated that half of the charge separation was impaired in F/RT, however, low-temperature EPR revealed the light-induced FX signal at a similar size as in the case of C/RT. This indicates that the two branches of electron transfer in PSI were affected differently. Electron transfer at the A-branch was inhibited in F/RT and also partially in C/LT, while the B-branch remained active.

Fluorescein analogues as photoremovable protecting groups absorbing at ∼520 nm.

A new photoremovable protecting group, (6-hydroxy-3-oxo-3H-xanthen-9-yl)methyl (1), with a molar absorption coefficient ε of ∼4 × 10(4) m(-1) cm(-1) at ∼520 nm for the release of carboxylates or phosphates is reported. Three derivatives of 1 (diethyl phosphate, acetate, and bromide) were isolated as complexes with DDQ and shown to release the ligands with quantum yields ≤2.4% in aqueous solution.

Multiscale Transient Absorption Study of the Fluorescent Protein Dreiklang and Two Point Variants Provides Insight into Photoswitching and Nonproductive Reaction Pathways.

Dreiklang is a reversibly photoswitchable fluorescent protein used as a probe in advanced fluorescence imaging. It undergoes a unique and still poorly understood photoswitching mechanism based on the reversible addition of a water molecule to the chromophore. We report the first comprehensive study of the dynamics of this reaction by transient absorption spectroscopy from 100 fs to seconds in the original Dreiklang protein and two point variants. The picture that emerges from our work is that of a competition between photoswitching and nonproductive reaction pathways. We found that photoswitching had a low quantum yield of 0.4%. It involves electron transfer from a tyrosine residue (Tyr203) to the chromophore and is completed in 33 ns. Nonproductive deactivation pathways comprise recombination of a charge transfer intermediate, excited-state proton transfer from the chromophore to a histidine residue (His145), and decay to the ground state via micro-/millisecond-lived intermediates.

Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase.

Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP (CvFAP) can convert n-octanoic acid in vitro four times faster than n-hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a CvFAP-based production rate over 10-fold higher for n-heptane than for n-pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that CvFAP's high catalytic activity on n-octanoic acid is, in part, due to an autocatalytic effect of its n-heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Light-activated cryptochrome reacts with molecular oxygen to form a flavin-superoxide radical pair consistent with magnetoreception.

Cryptochromes are flavin-based photoreceptors occurring throughout the biological kingdom, which regulate growth and development in plants and are involved in the entrainment of circadian rhythms of both plants and animals. A number of recent theoretical works suggest that cryptochromes might also be the receptors responsible for the sensing of the magnetic field of the earth (e.g. in insects, migratory birds, or migratory fish). Cryptochromes undergo forward light-induced reactions involving electron transfer to excited state flavin to generate radical intermediates, which correlate with biological activity. Here, we give evidence of a mechanism for the reverse reaction, namely dark reoxidation of protein-bound flavin in Arabidopsis thaliana cryptochrome (AtCRY1) by molecular oxygen that involves formation of a spin-correlated FADH(•)-superoxide radical pair. Formation of analogous radical pairs in animal cryptochromes might enable them to function as magnetoreceptors.

[Ru(bpy)(3)](2+) as a reference in transient absorption spectroscopy: differential absorption coefficients for formation of the long-lived (3)MLCT excited state.

Transient absorption spectroscopy and other time-resolved methods are commonly used to study chemical reactions and biological processes induced by absorption of light. In order to scale the signal amplitude or to compare results obtained under different conditions, it is advisable to use a reference system, a standard of convenient and well-defined properties. Finding Tris(bipyridine)ruthenium(ii), [Ru(bpy)(3)](2+), a suitable candidate for a transient-absorption spectroscopy reference due to its favourable photochemical properties, we have determined accurate relative values of differential molar absorption coefficients (Δε) for light-induced formation of the metal-to-ligand charge transfer (MLCT) excited triplet state at several relevant wavelengths (wavelengths of commercially available lasers) in the UV and visible regions. We have also attempted to determine the absolute value of Δε close to the wavelength of maximum bleaching (∼450 nm) and we propose to narrow down the interval of conceivable values for Δε(450) from the broad range of published values (-0.88 × 10(4) M(-1)cm(-1) to -1.36 × 10(4) M(-1)cm(-1)) to -1.1 × 10(4) M(-1)cm(-1)± 15%. Having ourselves successfully applied [Ru(bpy)(3)](2+) as a standard in a recent time-resolved study of enzymatic DNA repair, we would like to encourage other scientists to use this convenient tool as a reference in their future spectroscopic studies on time scales from picoseconds to hundreds of nanoseconds.

Limited solvation of an electron donating tryptophan stabilizes a photoinduced charge-separated state in plant (6-4) photolyase.

(6-4) Photolyases ((6-4) PLs) are ubiquitous photoenzymes that use the energy of sunlight to catalyze the repair of carcinogenic UV-induced DNA lesions, pyrimidine(6-4)pyrimidone photoproducts. To repair DNA, (6-4) PLs must first undergo so-called photoactivation, in which their excited flavin adenine dinucleotide (FAD) cofactor is reduced in one or two steps to catalytically active FADH- via a chain of three or four conserved tryptophan residues, transiently forming FAD•-/FADH- ⋯ TrpH•+ pairs separated by distances of 15 to 20 Å. Photolyases and related photoreceptors cryptochromes use a plethora of tricks to prevent charge recombination of photoinduced donor-acceptor pairs, such as chain branching and elongation, rapid deprotonation of TrpH•+ or protonation of FAD•-. Here, we address Arabidopsis thaliana (6-4) PL (At64) photoactivation by combining molecular biology, in vivo survival assays, static and time-resolved spectroscopy and computational methods. We conclude that At64 photoactivation is astonishingly efficient compared to related proteins-due to two factors: exceptionally low losses of photoinduced radical pairs through ultrafast recombination and prevention of solvent access to the terminal Trp3H•+, which significantly extends its lifetime. We propose that a highly conserved histidine residue adjacent to the 3rd Trp plays a key role in Trp3H•+ stabilization.

Historical ferrous slag induces modern environmental problems in the Moravian Karst (Czech Republic).

Ferrous slag produced by a historic smelter is washed from a slagheap and transported by a creek through a cave system. Slag filling cave spaces, abrasion of cave walls / calcite speleothems, and contamination of the aquatic environment with heavy metals and other toxic components are concerns. We characterize the slag in its deposition site, map its transport through the cave system, characterize the effect of slag transport, and evaluate the risks to both cave and aqueous environments. The study was based on chemical and phase analysis supported laboratory experiments and geochemical modeling. The slag in the slagheap was dominated by amorphous glass phase (66 to 99 wt%) with mean composition of 49.8 ± 2.8 wt% SiO2, 29.9 ± 1.6 wt% CaO, 13.4 ± 1.2 wt% Al2O3, 2.7 ± 0.3 wt% K2O, and 1.2 ± 0.1 wt% MgO. Minerals such as melilite, plagioclase, anorthite, and wollastonite / pseudowollastonite with lower amounts of quartz, cristobalite, and calcite were detected. Slag enriches the cave environment with Se, As, W, Y, U, Be, Cs, Sc, Cd, Hf, Ba, Th, Cr, Zr, Zn, and V. However, only Zr, V, Co, and As exceed the specified limits for soils (US EPA and EU limits). The dissolution lifetime of a 1 mm3 volume of slag was estimated to be 27,000 years, whereas the mean residence time of the slag in the cave is much shorter, defined by a flood frequency of ca. 47 years. Consequently, the extent of slag weathering and contamination of cave environment by slag weathering products is small under given conditions. However, slag enriched in U and Th can increase radon production as a result of alpha decay. The slag has an abrasive effect on surrounding rocks and disintegrated slag can contaminate calcite speleothems.

Electron relay race in peptides.

A peptide assay was developed that allows the measurement of electron-transfer (ET) efficiencies in peptides. It turns out that two-step ET processes are faster than single-step reactions. This requires relay amino acids with appropriate redox potentials. Not only aromatic but also sulfur-containing aliphatic amino acids can act as stepping stones for the charge. With tryptophan, histidine, and cysteine the reaction is a more complex proton-coupled ET.