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.     

Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo

Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor. The cofactor becomes oxidized to the FADH(*) blue neutral radical during purification. The E-FADH(*) form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306. It is thought that the E-FADH(*) form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH(*) that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair. In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo. We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate. We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.     

DNA repair mechanism by photolyase: electron transfer path from the photolyase catalytic cofactor FADH(-) to DNA thymine dimer

Photolyase is an enzyme that catalyses photorepair of thymine dimers in UV damaged DNA by electron transfer reaction. The structure of the photolyase/DNA complex is unknown at present. Using crystal structure coordinates of the substrate-free enzyme from E. coli, we have recently built a computer molecular model of a thymine dimer docked to photolyase catalytic site and studied molecular dynamics of the system. In this paper, we present analysis of the electronic coupling and electron transfer pathway between the catalytic cofactor FADH(-) and the pyrimidine dimer by the method of interatomic tunneling currents. Electronic structure is treated in the extended Hückel approximation. The root mean square transfer matrix element is about 6 cm(-1), which is consistent with the experimentally determined rate of transfer. We find that electron transfer mechanism responsible for the repair utilizes an unusual folded conformation of FADH(-) in photolyases, in which the isoalloxazine ring of the flavin and the adenine are in close proximity, and the peculiar features of the docked orientation of the dimer. The tunneling currents show explicitly that despite of the close proximity between the donor and acceptor complexes, the electron transfer mechanism between the flavin and the thymine bases is not direct, but indirect, with the adenine acting as an intermediate. These calculations confirm the previously made conclusion based on an indirect evidence for such mechanism.     

Fourier-transform infrared study of the photoactivation process of Xenopus (6-4) photolyase

Photolyases (PHRs) are blue light-activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The flavin adenine dinucleotide (FAD) chromophore of PHRs has four different redox states: oxidized (FAD(ox)), anion radical (FAD(?-)), neutral radical (FADH(?)), and fully reduced (FADH(-)). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV-visible spectroscopy to study the detailed photoactivation process of Xenopus (6-4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FAD(ox) is converted to semiquinone via light-induced one-electron and one-proton transfers and then to FADH(-) by light-induced one-electron transfer. We successfully trapped FAD(?-) at 200 K, where electron transfer occurs but proton transfer does not. UV-visible spectroscopy following 450 nm illumination of FAD(ox) at 277 K defined the FADH(?)/FADH(-) mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic C=O stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested by UV-visible and FTIR analysis of FAD(?-) at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's β-sheet during initial electron transfer (FAD(?-) formation), a transient increase in α-helicity during proton transfer (FADH(?) formation), and reversion to the initial amide I signal following subsequent electron transfer (FADH(-) formation). Consequently, in (6-4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH(-) did not perturb α-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of these FTIR observations.     

Potentiometric analysis of UDP-galactopyranose mutase: stabilization of the flavosemiquinone by substrate

UDP-galactopyranose mutase is a flavoprotein which catalyses the interconversion of UDP-galactopyranose and UDP-galactofuranose. The enzyme is of interest because it provides the activated biosynthetic precursor of galactofuranose, a key cell wall component of many bacterial pathogens. The reaction mechanism of this mutase is intriguing because the anomeric oxygen forms a glycosidic bond, which means that the reaction must proceed by a novel mechanism involving ring breakage and closure. The structure of the enzyme is known, but the mechanism, although speculated on, is not resolved. The overall reaction is electrically neutral but a crypto-redox reaction is suggested by the requirement that the flavin must adopt the reduced form for activity. Herein we report a thermodynamic analysis of the enzyme's flavin cofactor with the objective of defining the system and setting parameters for possible reaction schemes. The analysis shows that the neutral semiquinone (FADH(*)) is stabilized in the presence of substrate and the fully reduced flavin is the anionic FADH(-) rather than the neutral FADH(2). The anionic FADH(-) has the potential to act as a rapid 1-electron donor/acceptor without being slowed by a coupled proton transfer and is therefore an ideal crypto-redox cofactor.     

Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase.

DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox. The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2. Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V). Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2. Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH. is a better oxidant than FADH.. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer. This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2. The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2. Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy transfer in lactose repressor protein modified with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate

Energy transfer between the two tryptophan residues in the lactose repressor protein and the fluorescent moiety of the cysteine-specific reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (1,5-IAEDANS) has been examined. Modification of repressor with this compound did not affect operator or inducer binding. 1,5-IAEDANS reacted primarily with Cys140 in wild-type repressor [Schneider et al. (1984) Biochemistry 23, 2221]; in the presence of inducer, modification at Cys107 increased, while reaction at Cys140 remained unchanged. Energy transfer between tryptophans and the AEDANS moiety(ies) in wild-type lac repressor occurred with an efficiency of 6.7 +/- 1.9% in the absence and 7.8 +/- 1.6% in the presence of inducer. The distance between the Trp donor(s) and the acceptor in wild-type repressor was calculated to be in the range approximately 35 A under both conditions. The similarity in efficiency despite large differences in the amount of acceptor attached to Cys107 when inducer is bound indicates that the AEDANS group at position 107 does not participate significantly in energy transfer and that the label at position 140 acts as the primary acceptor group. The similarity of energy-transfer efficiency (7.1 +/- 3.8%) observed for 1,5-IAEDANS-modified monomeric mutant repressor (Y282D) indicates that the transfer is primarily intrasubunit in the native tetramer. Measurements using two mutant repressors (each with a single tryptophan and modified with 1,5-IAEDANS) demonstrated that both tryptophans can serve as donor in the energy-transfer process. The W201Y repressor (containing Trp220) exhibited a transfer efficiency lower than wild type (5.6 +/- 2.4%), corresponding to a slightly larger distance between the donor-acceptor pair in this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an "inverse" deuterium isotope effect

We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH(-) and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (k(H)/k(D)=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH(-) (electron transfer time constant tau(et) in the order of 10-50 micros), and one where electron transfer from FADH(-) (tau(et)=25 ms) is followed by reprotonation of the W306 anion.     

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human neuronal nitric-oxide synthase and inducible nitric-oxide synthase flavin domains

Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.     

Metabolomic analyses show that electron donor and acceptor ratios control anaerobic electron transfer pathways in Shewanella oneidensis

This study investigated the physiological impact of changing electron donor–acceptor ratios on electron transfer pathways in the metabolically flexible subsurface bacterium Shewanella oneidensis, using batch and chemostat cultures, with an azo dye (ramazol black B) as the model electron acceptor. Altering the growth rate did result in changes in biomass yield, but not in other key physiological parameters including the total cytochrome content of the cells, the production of extracellular flavin redox shuttles or the potential of the organism to reduce the azo dye. Dramatic increases in the ability to reduce the dye were noted when cells were grown under conditions of electron acceptor (fumarate) limitation, although the yields of extracellular redox mediators (flavins) were similar under conditions of electron donor (lactate) or acceptor limitation. FT-IR spectroscopy confirmed shifts in the metabolic fingerprints of cells grown under these contrasting conditions, while spectrophotometric analyses supported a critical role for c-type cytochromes, expressed at maximal concentrations under conditions of electron acceptor limitation. Finally, key intracellular metabolites were quantified in batch experiments at various electron donor and acceptor ratios and analysed using discriminant analysis and a Bayesian network to construct a central metabolic pathway model for cells grown under conditions of electron donor or acceptor limitation. These results have identified key mechanisms involved in controlling electron transfer in Shewanella species, and have highlighted strategies to maximise reductive activity for a range of bioprocesses.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways

Cryptochromes are blue light receptors with multiple signaling roles in plants and animals. Plant cryptochrome (cry1 and cry2) biological activity has been linked to flavin photoreduction via an electron transport chain comprising three evolutionarily conserved tryptophan residues known as the Trp triad. Recently, it has been reported that cry2 Trp triad mutants, which fail to undergo photoreduction in vitro, nonetheless show biological activity in vivo, raising the possibility of alternate signaling pathways. Here, we show that Arabidopsis thaliana cry2 proteins containing Trp triad mutations indeed undergo robust photoreduction in living cultured insect cells. UV/Vis and electron paramagnetic resonance spectroscopy resolves the discrepancy between in vivo and in vitro photochemical activity, as small metabolites, including NADPH, NADH, and ATP, were found to promote cry photoreduction even in mutants lacking the classic Trp triad electron transfer chain. These metabolites facilitate alternate electron transfer pathways and increase light-induced radical pair formation. We conclude that cryptochrome activation is consistent with a mechanism of light-induced electron transfer followed by flavin photoreduction in vivo. We further conclude that in vivo modulation by cellular compounds represents a feature of the cryptochrome signaling mechanism that has important consequences for light responsivity and activation.     

Mechanism of the highly efficient quenching of tryptophan fluorescence in human gammaD-crystallin

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.     

Discrimination of class I cyclobutane pyrimidine dimer photolyase from blue light photoreceptors by single methionine residue

DNA photolyase recognizes ultraviolet-damaged DNA and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. Theoretical analysis of the electron-tunneling pathways of the DNA photolyase derived from Anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. Here, we report the unexpectedly important role of the single methionine residue, Met-353, where busy trafficking of electron-tunneling currents is observed. The amino acid conservation pattern of Met-353 in the homologous sequences perfectly correlates with experimentally verified annotation as photolyases. The bioinformatics sequence analysis also suggests that the residue plays a pivotal role in biological function. Consistent findings from different disciplines of computational biology strongly suggest the pivotal role of Met-353 in the biological function of DNA photolyase.     

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.     

Roles of FAD and 8-hydroxy-5-deazaflavin chromophores in photoreactivation by Anacystis nidulans DNA photolyase.

DNA photolyase from the cyanobacterium Anacystis nidulans contains two chromophores, flavin adenine dinucleotide (FADH2) and 8-hydroxy-5-deazaflavin (8-HDF) (Eker, A. P. M., Kooiman, P., Hessels, J. K. C., and Yasui, A. (1990) J. Biol. Chem. 265, 8009-8015). While evidence exists that the flavin chromophore (in FADH2 form) can catalyze photorepair directly and that the 8-HDF chromophore is the major photosensitizer in photoreactivation it was not known whether 8-HDF splits pyrimidine dimer directly or indirectly through energy transfer to FADH2 at the catalytic center. We constructed a plasmid which over-produces the A. nidulans photolyase in Escherichia coli and purified the enzyme from this organism. Apoenzyme was prepared and enzyme containing stoichiometric amounts of either or both chromophores was reconstituted. The substrate binding and catalytic activities of the apoenzyme (apoE), E-FADH2, E-8-HDF, E-FAD(ox)-8-HDF, and E-FADH2-8-HDF were investigated. We found that FAD is required for substrate binding and catalysis and that 8-HDF is not essential for binding DNA, and participates in catalysis only through energy transfer to FADH2. The quantum yields of energy transfer from 8-HDF to FADH2 and of electron transfer from FADH2 to thymine dimer are near unity.     

Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase.

The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation and function of flavin anion radical in cryptochrome 1 blue-light photoreceptor of monarch butterfly

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.     

Analysis of amino acid residues involved in catalysis of polyethylene glycol dehydrogenase from Sphingopyxis terrae, using three-dimensional molecular modeling-based kinetic characterization of mutants

Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q10 was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.     

A new classification of the amino acid side chains based on doublet acceptor energy levels

We describe a new classification of the amino acid side chains based on the potential energy level at which each will accept an extra (doublet) electron. The doublet acceptor energy level, and the doublet acceptor orbital were calculated using semiempirical INDO/2-UHF molecular orbital theory. The results of these calculations show that the side chains fall into four groups. We have termed these groups repulsive, insulating, semiconducting, and attractive in accordance with where each lies on the relative energy scale. We use this classification to examine the role of residues between the donor and acceptor in modulating the rate and mechanism of electron transfer in proteins. With the calculated acceptor levels, we construct a potential barrier for those residues between the donor and acceptor. It is the area beneath this barrier that determines the decay of electronic coupling between donor and acceptor, and thus the transfer rate. We have used this schematic approach to characterize the four electron transfer pathways in myoglobin recently studied by Mayo et al. (Mayo, S.L., W.R. Ellis, R.J. Crutchley, and H.B. Gray. 1986. Science [Wash. DC]. 233:948-952).