Structural Changes during the Photorepair and Binding Processes of Xenopus (6-4) Photolyase with (6-4) Photoproducts in Single- and Double-Stranded DNA.

Photolyases (PHRs) repair ultraviolet (UV)-induced DNA photoproducts into normal bases. In this study, we measured the conformational changes upon photoactivation and photorepair processes of a PHR and its specific substrates, (6-4)PHR and a pyrimidine(6-4)pyrimidone photoproduct ((6-4)PP), by light-induced difference Fourier transform infrared (FT-IR) spectroscopy. The single-stranded DNA with (6-4)PP (ss(6-4)PP) was used as a substrate and the resultant FT-IR spectra were compared with the previous results on double-stranded DNA with (6-4)PP (ds(6-4)PP). In the excess amount of substrate to the enzyme, different ss(6-4)PP photorepair FT-IR signals were obtained in an illumination time-dependent manner. As reported for ds(6-4)PP, the early stages of the photoreaction involve the changes in the ss(6-4)PP only, while the late stages of the reaction involve the ss(6-4)PP repair-associated changes and dissociation from (6-4)PHR. From these spectra, difference spectra originating from the binding/dissociation spectrum were extracted. The signals of the C═O stretches of (6-4)PP and repaired thymines in the single- and double-stranded DNA were tentatively assigned. The C═O stretches of (6-4)PP were observed at frequencies that reflect single- and double-stranded DNA environments in aqueous solution, reflecting the different hydrogen-bonding environments. The conformational changes of PHR upon binding of ss(6-4)PP and ds(6-4)PP were similar, suggesting that the conformational change is limited to the (6-4)PP binding pocket region. We interpreted that ds(6-4)PP may be bound together without any special mechanism for flipping out.

ATP binding promotes light-induced structural changes to the protein moiety of Arabidopsis cryptochrome 1.

Cryptochromes (CRYs) are blue-light receptors involved in photomorphogenesis in plants. Flavin adenine dinucleotide (FAD) is one of the chromophores of cryptochromes; its resting state oxidized form is converted into a signalling state neutral semiquionod radical (FADH˙) form. Studies have shown that cryptochrome 1 from Arabidopsis thaliana (AtCRY1) can bind ATP at its photolyase homology region (PHR), resulting in accumulation of FADH˙ form. This study used light-induced difference Fourier transform infrared spectroscopy to investigate how ATP influences structural changes in AtCRY1-PHR during the photoreaction. In the presence of ATP, there were large changes in the signals from the protein backbone compared with in the absence of ATP. The deprotonation of a carboxylic acid was observed only in the presence of ATP; this was assigned as aspartic acid (Asp) 396 through measurement of Asp to glutamic acid mutants. This corresponds to the protonation state of Asp396 estimated from the reported pKa values of Asp396; that is, the side chain of Asp396 is deprotonated and protonated for the ATP-free and -bound forms, respectively, in our experimental condition at pH8. Therefore, Asp396 acts a proton donor to FAD when it is ptotonated. It was indicated that the protonation/deprotination process of Asp396 is correlated with the accunumulation of FADH˙ and protein conformational changes.

Self-Assembly of the Cephalopod Protein Reflectin.

Films from the cephalopod protein reflectin demonstrate multifaceted functionality as infrared camouflage coatings, proton transport media, and substrates for growth of neural stem cells. A detailed study of the in vitro formation, structural characteristics, and stimulus response of such films is presented. The reported observations hold implications for the design and development of advanced cephalopod-inspired functional materials.

Functional Conversion of CPD and (6-4) Photolyases by Mutation.

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.

Single Hydrogen Bond Donation from Flavin N5 to Proximal Asparagine Ensures FAD Reduction in DNA Photolyase.

The spread of the absorbance of the stable FADH(•) radical (300-700 nm) allows CPD photolyase to highly efficiently form FADH(-), making it functional for DNA repair. In this study, FTIR spectroscopy detected a strong hydrogen bond, from FAD N5-H to the carbonyl group of the Asn378 side chain, that is modulated by the redox state of FAD. The observed characteristic frequency shifts were reproduced in quantum-mechanical models of the flavin binding site, which were then employed to elucidate redox tuning governed by Asn378. We demonstrate that enhanced hydrogen bonding of the Asn378 side chain with the FADH(•) radical increases thermodynamic stabilization of the radical state, and further ensures kinetic stabilization and accumulation of the fully reduced FADH(-) state.

Structural Changes of the Active Center during the Photoactivation of Xenopus (6-4) Photolyase.

Photolyases (PHRs) repair the UV-induced photoproducts, cyclobutane pyrimidine dimer (CPD) or pyrimidine-pyrimidone (6-4) photoproduct [(6-4) PP], restoring normal bases to maintain genetic integrity. CPD and (6-4) PP are repaired by substrate-specific PHRs, CPD PHR and (6-4) PHR, respectively. Flavin adenine dinucleotide (FAD) is the chromophore of both PHRs, and the resting oxidized form (FAD(ox)), at least under in vitro purified conditions, is first photoconverted to the neutral semiquinoid radical (FADH(•)) form, followed by photoconversion into the enzymatically active fully reduced (FADH(-)) form. Previously, we reported light-induced difference Fourier transform infrared (FTIR) spectra corresponding to the photoactivation process of Xenopus (6-4) PHR. Spectral differences between the absence and presence of (6-4) PP were observed in the photoactivation process. To identify the FTIR signals where these differences appeared, we compared the FTIR spectra of photoactivation (i) in the presence and absence of (6-4) PP, (ii) of (13)C labeling, (15)N labeling, and [(14)N]His/(15)N labeling, and (iii) of H354A and H358A mutants. We successfully assigned the vibrational bands for (6-4) PP, the α-helix and neutral His residue(s). In particular, we assigned three bands to the C ═ O groups of (6-4) PP in the three different redox states of FAD. Furthermore, the changed hydrogen bonding environments of C ═ O groups of (6-4) PP suggested restructuring of the binding pocket of the DNA lesion in the process of photoactivation.

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus.

UNLABELLED: Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCE: By protecting microbes against reactive oxygen insults, SODs aid survival of many bacteria within their hosts. Despite the ubiquity and conservation of these key enzymes, notable species-specific differences relevant to pathogenesis remain undefined. To probe mechanisms that govern the functioning of Neisseria meningitidis and Brucella abortus SODs, we used X-ray structures, enzymology, modeling, and murine infection experiments. We identified virulence determinants common to the two homologs, assembly differences, and a unique metal reservoir within meningococcal SOD that stabilizes the enzyme and may provide a safeguard against copper toxicity. The insights reported here provide a rationale and a basis for SOD-specific drug design and an extension of immunogen design to target two important pathogens that continue to pose global health threats.

Reaction dynamics of the UV-B photosensor UVR8.

UVR8 is a recently discovered ultraviolet-B (UV-B) photoreceptor protein identified in plants and algae. In the dark state, UVR8 exists as a homodimer, whereas UV-B irradiation induces UVR8 monomerization and initiation of signaling. Although the biological functions of UVR8 have been studied, the fundamental reaction mechanism and associated kinetics have not yet been fully elucidated. Here, we used the transient grating method to determine the reaction dynamics of UVR8 monomerization based on its diffusion coefficient. We found that the UVR8 photodissociation reaction proceeds in three stages: (i) photoexcitation of cross-dimer tryptophan (Trp) pyramids; (ii) an initial conformational change with a time constant of 50 ms; and (iii) dimer dissociation with a time constant of 200 ms. We identified W285 as the key Trp residue responsible for initiating this photoreaction. Although the C-terminus of UVR8 is essential for biological interactions and signaling via downstream components such as COP1, no obvious differences were detected between the photoreactions of wild-type UVR8 (amino acids 1-440) and a mutant lacking the C-terminus (amino acids 1-383). This similarity indicates that the conformational change associated with stage ii cannot primarily be attributed to this region. A UV-B-driven conformational change with a time constant of 50 ms was also detected in the monomeric mutants of UVR8. Dimer recovery following monomerization, as measured by circular dichroism spectroscopy, was decreased under oxygen-purged conditions, suggesting that redox reactivity is a key factor contributing to the UVR8 oligomeric state.

FTIR study of CPD photolyase with substrate in single strand DNA.

Photolyases (PHRs) utilize near UV/blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer (CPD)-PHR binds flavin adenine dinucleotide (FAD) as a cofactor and repairs CPD lesions in double-stranded DNA. To understand the activation and repair mechanism of CPD-PHR, we applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to CPD-PHR, whose signals were identified by use of isotope-labeling. To further investigate the enzymatic function, here we study the activation and repair mechanism of CPD-PHR with the substrate in single strand DNA, and the obtained FTIR spectra are compared with those in double-stranded DNA, the natural substrate. The difference spectra of photoactivation, the fully-reduced (FADH(-)) minus semiquinone (FADH(•)) spectra, are almost identical in the presence of single strand and double-stranded DNA, except for slight spectral modification in the amide-I region. On the other hand, the difference spectra of photorepair were highly substrate dependent. Strong bands of the C=O stretch (1,720-1,690 cm(-1)) and phosphate vibrations (1,090-1,060 cm(-1)) of double-stranded DNA may have disappeared in the case of single strand DNA. However, an isotope-labeled enzyme study revealed that spectral features upon DNA repair are similar between both substrates, and the main reason for the apparent spectral difference originates from structural flexibility of DNA after repair.

Structural role of two histidines in the (6-4) photolyase reaction.

Photolyases (PHRs) are DNA repair enzymes that revert UV-induced photoproducts, either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts (PPs), into normal bases to maintain genetic integrity. (6-4) PHR must catalyze not only covalent bond cleavage, but also hydroxyl or amino group transfer, yielding a more complex mechanism than that postulated for CPD PHR. Previous mutation analysis revealed the importance of two histidines in the active center, H354 and H358 for Xenopus (6-4) PHR, whose mutations significantly lowered the enzymatic activity. Based upon highly sensitive FTIR analysis of the repair function, here we report that both H354A and H358A mutants of Xenopus (6-4) PHR still maintain their repair activity, although the efficiency is much lower than that of the wild type. Similar difference FTIR spectra between the wild type and mutant proteins suggest a common mechanism of repair in which (6-4) PP binds to the active center of each mutant, and is released after repair, as occurs in the wild type. Similar FTIR spectra also suggest that a decrease in volume by the H-to-A mutation is possibly compensated by the addition of water molecule( s). Such a modified environment is sufficient for the repair function that is probably controlled by proton-coupled electron transfer between the enzyme and substrate. On the other hand, two histidines must work in a concerted manner in the active center of the wild-type enzyme, which significantly raises the repair efficiency.

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes.

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.

Copper-based pulsed dipolar ESR spectroscopy as a probe of protein conformation linked to disease states.

We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu(2+) ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.

Flavin adenine dinucleotide chromophore charge controls the conformation of cyclobutane pyrimidine dimer photolyase α-helices.

Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon-chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure-function study by FTIR spectroscopy of the Escherichia coli cyclobutane pyrimidine dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which revealed protein dynamics for both processes beyond simple one-electron reduction and ejection, respectively. The FTIR data suggest that the synergistic cofactor-protein partnership in CPD-PHR linked to changes in the shape of FAD upon one-electron reduction may be coordinated with conformational changes in the apoprotein, allowing it to fit the DNA substrate. Activation of the CPD-PHR chromophore primes the apoprotein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apoprotein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues.

ATP binding turns plant cryptochrome into an efficient natural photoswitch.

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction.

FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis.

Abscisic acid (ABA) is a plant hormone that regulates plant growth and development and mediates abiotic stress responses. Direct cellular monitoring of dynamic ABA concentration changes in response to environmental cues is essential for understanding ABA action. We have developed ABAleons: ABA-specific optogenetic reporters that instantaneously convert the phytohormone-triggered interaction of ABA receptors with PP2C-type phosphatases to send a fluorescence resonance energy transfer (FRET) signal in response to ABA. We report the design, engineering and use of ABAleons with ABA affinities in the range of 100-600 nM to map ABA concentration changes in plant tissues with spatial and temporal resolution. High ABAleon expression can partially repress Arabidopsis ABA responses. ABAleons report ABA concentration differences in distinct cell types, ABA concentration increases in response to low humidity and NaCl in guard cells and to NaCl and osmotic stress in roots and ABA transport from the hypocotyl to the shoot and root. DOI: http://dx.doi.org/10.7554/eLife.01739.001.

Variable electron transfer pathways in an amphibian cryptochrome: tryptophan versus tyrosine-based radical pairs.

Electron transfer reactions play vital roles in many biological processes. Very often the transfer of charge(s) proceeds stepwise over large distances involving several amino acid residues. By using time-resolved electron paramagnetic resonance and optical spectroscopy, we have studied the mechanism of light-induced reduction of the FAD cofactor of cryptochrome/photolyase family proteins. In this study, we demonstrate that electron abstraction from a nearby amino acid by the excited FAD triggers further electron transfer steps even if the conserved chain of three tryptophans, known to be an effective electron transfer pathway in these proteins, is blocked. Furthermore, we were able to characterize this secondary electron transfer pathway and identify the amino acid partner of the resulting flavin-amino acid radical pair as a tyrosine located at the protein surface. This alternative electron transfer pathway could explain why interrupting the conserved tryptophan triad does not necessarily alter photoreactions of cryptochromes in vivo. Taken together, our results demonstrate that light-induced electron transfer is a robust property of cryptochromes and more intricate than commonly anticipated.

Detection of distinct α-helical rearrangements of cyclobutane pyrimidine dimer photolyase upon substrate binding by Fourier transform infrared spectroscopy.

Photolyases (PHRs) utilize near-ultraviolet (UV)-blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second photolyase known as (6-4) PHR, present in a range of higher organisms, uniquely repairs (6-4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6-4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6-4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR involves the transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the dimers to maintain the DNA's integrity. Here, we measured and analyzed difference FTIR spectra for the photoactivation and DNA photorepair processes of CPD-PHR. We identified light-dependent signals only in the presence of substrate. The signals, presumably arising from a protonated carboxylic acid or the DNA substrate, implicate conformational rearrangements of the protein and substrate during the repair process. Deuterium exchange FTIR measurements of CPD-PHR highlight potential differences in the photoactivation and photorepair mechanisms in comparison to those of (6-4) PHR. Although CPD-PHR and (6-4) PHR appear to exhibit similar overall structures, our studies indicate that distinct conformational rearrangements, especially in the α-helices, are initiated within these enzymes upon binding of their respective DNA substrates.

Amyotrophic lateral sclerosis: update and new developments.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease. It is typically characterized by adult-onset degeneration of the upper and lower motor neurons, and is usually fatal within a few years of onset. A subset of ALS patients has an inherited form of the disease, and a few of the known mutant genes identified in familial cases have also been found in sporadic forms of ALS. Precisely how the diverse ALS-linked gene products dictate the course of the disease, resulting in compromised voluntary muscular ability, is not entirely known. This review addresses the major advances that are being made in our understanding of the molecular mechanisms giving rise to the disease, which may eventually translate into new treatment options.

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 ß-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.