Use of flavin photochemistry to probe intraprotein and interprotein electron transfer mechanisms

Photoexcitation of flavin analogs generates the lowest triplet state (via intersystem crossing from the first excited singlet state) in the nanosecond time domain and with high quantum efficiency. The triplet, being a strong oxidant, can abstract a hydrogen atom (or an electron) from a reduced donor in a diffusion-controlled reaction. If the donor is a redox protein, the oxidation process can be used to initiate an electron transfer sequence involving either intramolecular or intermolecular reactions. If the donor is an organic compound such as EDTA, the neutral flavin semiquinone will be produced by H atom abstraction; this is a strong reductant and can subsequently transfer a hydrogen atom (or an electron) to an oxidized redox protein, thereby again initiating a sequence of intramolecular or intermolecular processes. If flavin photoexcitation is accomplished using a pulsed laser light source, the initiation of these protein electron transfer reactions can be made to occur in the nanosecond to microsecond time domain, and the sequence of events can be followed by time-resolved spectrophotometry to obtain rate constants and thus mechanistic information. The present paper describes this technology, and selected examples of its use in the investigation of redox protein mechanisms are given.     

Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees . The catalytically potent state FADH- can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees , in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH degrees * <-- W382 <-- W359 <-- W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH degrees and leads to the formation of FADH- and a deprotonated tryptophanyl radical, most likely W359 degrees. These photoproducts are formed in less than 10 ns and recombine to the dark state in approximately 1 micros. These results support the electron hopping mechanism.     

Decarboxylation is a significant reaction pathway for photolabile calcium chelators and related compounds.

Photolysis of amino acids that bear a 2-nitrobenzyl protecting group on the amino nitrogen involves a normal 2-nitrobenzyl-type photocleavage to release the amino acid but also a second mechanistic pathway, that appears to be initiated by single electron transfer from the amino group to the excited state of the nitroaromatic. The end result of this pathway is photodecarboxylation. Quantitative experiments suggest that this latter pathway can contribute between 10 and 80% of the total reaction flux in different compounds. It appears that the aminium radical, the first product of the single electron transfer, can be intercepted by certain amino acids, resulting in a transfer of decarboxylation to this "sacrificial" amino acid. Possible implications for precise details of calcium release from photolabile derivatives of EDTA and EGTA are discussed.     

Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors

Photoinduced electron transfer in biological systems, especially in proteins, is a highly intriguing matter. Its mechanistic details cannot be addressed by structural data obtained by crystallography alone because this provides only static information on a given redox system. In combination with transient spectroscopy and site-directed manipulation of the protein, however, a dynamic molecular picture of the ET process may be obtained. In BLUF (blue light sensors using FAD) photoreceptors, proton-coupled electron transfer between a tyrosine and the flavin cofactor is the key reaction to switch from a dark-adapted to a light-adapted state, which corresponds to the biological signaling state. Particularly puzzling is the fact that, although the various naturally occurring BLUF domains show little difference in the amino acid composition of the flavin binding pocket, the reaction rates of the forward reaction differ quite largely from a few ps up to several hundred ps. In this study, we modified the redox potential of the flavin/tyrosine redox pair by site-directed mutagenesis close to the flavin C2 carbonyl and fluorination of the tyrosine, respectively. We provide information on how changes in the redox potential of either reaction partner significantly influence photoinduced proton-coupled electron transfer. The altered redox potentials allowed us furthermore to experimentally describe an excited state charge transfer intermediately prior to electron transfer in the BLUF photocycle. Additionally, we show that the electron transfer rate directly correlates with the quantum yield of signaling state formation.     

Flash photolysis of misonidazole and metronidazole

Laser flash photolysis at 355 nm of misonidazole or metronidazole in aqueous solutions produced the relatively long-lived nitro radical anion as the only observable transient species. When 266 nm excitation was used, a small yield of solvated electron was observed. It is suggested that the nitroimidazole first undergoes photoionization and the photoelectrons are scavenged by ground state nitroimidazole molecules to produce the nitro radical anion. Alternatively, added EDTA or carbonate ion acted as an electron donor to the excited state nitroimidazole molecule, thereby increasing the yield of nitro radical anion. The transient yield from metronidazole was about half that from misonidazole, while the phosphorescence intensity of metronidazole in an ethanol glass was about 20 times that of misonidazole. The misonidazole n, pi* triplet state is more easily reduced than that of metronidazole and, in the presence of an electron donor, the radical anion is postulated to result from electron transfer to the triplet state of the nitroimidazole.     

Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 2. Kinetic studies on the intramolecular electron transfer in trimethylamine and dimethylamine dehydrogenase

E.p.r. spectroscopy of the trimethylamine and dimethylamine dehydrogenases of Hyphomicrobium X indicates that the substrate-reduced forms of these enzymes exist in the triplet state, which arise through interaction of a reduced [4Fe-4S] cluster and flavosemiquinone, with e.p.r. signals which differ in detail from those of the trimethylamine dehydrogenase of bacterium W3A1. Under certain conditions the intramolecular electron transfer between the flavoquinol form of 6-S-cysteinyl-FMN and the [4Fe-4S] cluster in all three dehydrogenases was much slower than the preceding reduction of the flavin to the flavoquinol form. Trimethylamine dehydrogenases from both organisms show a time-dependent broadening of the e.p.r. signals centred around g = 2 after mixing with trimethylamine. The broadening of the e.p.r. signals could be correlated with an unexpected dependence of the rate of formation of the triplet state on substrate concentration. A model which accounts in a qualitative manner for the substrate dependence of the formation of the triplet state in the trimethylamine dehydrogenase of Hyphomicrobium X is proposed. The binding of the substrate to the reduced form of the enzyme seems to result in a conformational change of the enzyme to a form in which the rate of intramolecular electron transfer is decreased. This finding may be correlated with the observation of hyperbolic substrate inhibition for both trimethylamine dehydrogenases. The results indicate the transfer of an electron to the [4Fe-4S] cluster to be an obligatory step in catalysis and suggest that the transfer of electrons from these enzymes to electron acceptors is mediated solely through the [4Fe-4S] cluster.     

Effect of xenon on the excited States of phototropic receptor flavin in corn seedlings

The chemically inert, water-soluble heavy atom gas, xenon, at millimolar concentrations specifically quenches the triplet excited state of flavin in solution without quenching the flavin singlet excited state. The preferential quenching of the flavin triplet over the singlet excited state by Xe has been established by showing that the flavin triplet-sensitized photooxidation of NADH is inhibited while the fluorescence intensity and lifetime of flavin are not affected by Xe.     

Photochemical properties of Escherichia coli DNA photolyase: selective photodecomposition of the second chromophore

Escherichia coli DNA photolyase contains a stable flavin radical and a second chromophore (SC) of unknown structure. The effects of flash (both conventional and laser) excitation of either the radical alone or both the radical and the second chromophore have been investigated by variation of the excitation wavelengths. Radical excitation leads to an electron abstraction by the lowest excited doublet state of the radical from an amino acid residue, probably a cysteine or tyrosine. On a longer time scale, a back-reaction occurs that can be prevented by the presence of certain electron donors, e.g., thiols, NADH, or tyrosine, but not pyrimidine dimers. Excitation of the second chromophore leads to electronic energy transfer from second chromophore excited states to the ground-state flavin radical doublet state, thus increasing the population of the lowest excited doublet state. Repetitive excitation of the enzyme with white light leads to photodecomposition of the second chromophore but not of the flavin adenine dinucleotide cofactor. Enzyme with photodecomposed SC retains full activity.     

The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.     

Light-mediated reduction of flavoproteins with flavins as catalysts

It has been found that small amounts of free flavins greatly accelerate the photochemical reduction of flavoproteins both to the radical and fully reduced oxidation states. This catalytic effect has been shown to be due to the rapid photochemical reduction of the free flavin to its fully reduced state, followed by its reaction with the flavoprotein to yield flavoprotein radical and by its reaction with flavoprotein radical to yield fully reduced flavoprotein. Evidence is presented that the same route may occur with flavoproteins in the absence of added flavins. In this case the photoreduction is mediated by the small equilibrium concentration of free flavin coenzyme present in a flavorprotein solution. Hence, it is suggested that flavoprotein reduction with EDTA as photosubstrate does not involve an excited state of the holoprotein, nor contact of EDTA with the enzyme, but exchange of electrons between enzyme flavin and free reduced flavin.     

The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain

Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP(+) with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP(+) dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP(+)-bound state as compared with wild type. We conclude that Phe(1395) regulates nNOSr catalysis in two ways. It facilitates NADP(+) release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.     

Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.     

Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.     

Cellular metabolites modulate in vivo signaling of Arabidopsis cryptochrome-1

Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the ‘Trp triad.’ Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH°) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.     

Effects of amino acid replacements in yeast iso-1 cytochrome c on heme accessibility and intracomplex electron transfer in complexes with cytochrome c peroxidase

The kinetics of reduction of wild type and several site-specific mutants of yeast iso-1 cytochrome c (Arg-13----Ile, Gln-16----Ser, Gln-16----Lys, Lys-27----Gln, Lys-72----Asp), both free and in 1:1 complexes with yeast cytochrome c peroxidase, by free flavin semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H2O2-oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme within the electrostatically stabilized complex and the rate constants for intramolecular electron transfer at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Importantly, replacement by uncharged amino acids of Arg or Lys residues thought to be important in orientation and/or stabilization of the electron-transfer complex resulted in increased rates of electron transfer. In all cases, an increase in ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilized 1:1 complex is not optimized for electron transfer and that by neutralization of key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can orient themselves to allow the formation of a more efficient electron-transfer complex.     

Energy transfer from enzymically generated triplet carbonyl compounds to the fluorescent state of flavins

Enzymically generated triplet carbonyl compounds transfer energy to the fluorescent state of flavins as shown by the suppression of the carbonyl chemiphosphorescence and concomitant appearance of the flavin fluorescence. A Stern-Volmer analysis including the effect of the collisional quenching by a diene indicates that the transfer occurs by a long range process.The present results open the way to “photobiology without light”.     

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.     

Bacterial sarcosine oxidase: identification of novel substrates and a biradical reaction intermediate

Corynebacterial sarcosine oxidase contains both covalently and noncovalently bound FAD and forms complexes with various heterocyclic carboxylic acids (D-proline and 2-furoic, 2-pyrrolecarboxylic, and 2-thiophenecarboxylic acids). 2-Furoic acid, a competitive inhibitor with respect to sarcosine, selectively perturbs the absorption spectrum of the noncovalent flavin, suggesting that the enzyme has a single sarcosine binding site near the noncovalent flavin. Several heterocyclic amines have been identified as new substrates for the enzyme. Similar reactivity is observed with L-proline and L-pipecolic acid whereas L-2-azetidine-carboxylic acid is less reactive. Turnover with L-proline is slow (TN = 4.4 min-1) as compared with sarcosine (TN = 1000 min-1). Anaerobic reduction of the enzyme with heterocyclic amine substrates at pH 8.0 occurs as a biphasic reaction. A similar long-wavelength intermediate is formed in the initial fast phase of each reaction and then decays in a slower second phase to yield 1,5-dihydroFAD. The slow phase is not kinetically significant during aerobic turnover at pH 8.0 and is absent when the anaerobic reactions are conducted at pH 7.0. EPR and other studies at pH 7.0 show that the long-wavelength species is a half-reduced form of the enzyme (1 electron/substrate-reducible flavin) containing 0.9 mol of flavin radical/mol of substrate-reducible flavin. This biradical intermediate exhibits an absorption spectrum similar to that expected for a 50:50 mixture of red anionic and blue neutral flavin radicals. A similar long-wavelength species is observed during titration of the enzyme with sarcosine and other reductants. Studies with L-proline suggest that reduction of the enzyme involves initial transfer of two electrons to the noncovalent flavin. The covalent flavin is not required and can be complexed with sulfite without affecting the rate of electron transfer. The initial half-reduced form of the enzyme appears to be rapidly converted to the biradical form via comproportionation of the reduced noncovalent flavin with the oxidized covalent flavin.     

Fate of flavins in sensitized photodegradation of isohumulones and reduced derivatives: studies on formation of radicals via EPR combined with detailed product analyses.

Photodegradation of isohumulones accounts for formation of the lightstruck flavor in beer. The reactions involved are mediated by riboflavin, a natural photosensitizer present in beer in ppb quantities. The results of an investigation of this sensitized degradation process are presented herein. Product analyses and electron paramagnetic resonance spectroscopy, in steady-state as well as in time-resolved mode, offer extensive insight into the photophysical and photochemical details of the degradation mechanism. In contrast to energy transfer and Norrish type I alpha-cleavage reactions that take place on direct irradiation of isohumulones, the sensitization pathway proceeds via one-electron redox chemistry involving the excited triplet state of riboflavin and derivatives. The flavin semiquinone radical thus formed could be readily detected, either by steady state or by time-resolved electron paramagnetic resonance spectroscopy. Superimposed signals in the spectra revealed the presence of radical fragments derived from isohumulones or tetrahydroisohumulones, which, on recombination with riboflavin semiquinone radicals, produced stable reaction products that were identified by HPLC-MS. However, no superimposed signals were observed on sensitized irradiation of dihydroisohumulones.     

Origin of the transient electron paramagnetic resonance signals in DNA photolyase

DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron-oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.