Ya Ting Kao

Ultrafast dynamics of flavins in five redox states.

We report here our systematic studies of excited-state dynamics of two common flavin molecules, FMN and FAD, in five redox states--oxidized form, neutral and anionic semiquinones, and neutral and anionic fully reduced hydroquinones--in solution and in inert protein environments with femtosecond resolution. Using protein environments, we were able to stabilize two semiquinone radicals and thus observed their weak emission spectra. Significantly, we observed a strong correlation between their excited-state dynamics and the planarity of their flavin isoalloxazine ring. For a bent ring structure, we observed ultrafast dynamics from a few to hundreds of picoseconds and strong excitation-wavelength dependence of emission spectra, indicating deactivation during relaxation. A butterfly bending motion is invoked to get access to conical intersection(s) to facilitate deactivation. These states include the anionic semiquinone radical and fully reduced neutral and anionic hydroquinones in solution. In a planar configuration, flavins have a long lifetime of nanoseconds, except for the stacked conformation of FAD, where intramolecular electron transfer between the ring and the adenine moiety in 5-9 ps as well as subsequent charge recombination in 30-40 ps were observed. These observed distinct dynamics, controlled by the flavin ring flexibility, are fundamental to flavoenzymes functions, as observed in photolyase with a planar structure to lengthen the lifetime to maximize DNA repair efficiency and in insect type 1 cryptochrome with a flexible structure to vary the excited-state deactivation to modulate the functional channel.

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme’s high repair efficiency.

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD(*-)) and hydroquinone (FADH(-)) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH(-)*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD(*-)*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.

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, FADox, form. Exposure of the purified protein to light converts the FADox to the batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{FAD}^{{bar{{cdot}}}}) end{document} 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 FADox form in vivo and that, most likely, the batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{FAD}_{mathrm{ox}}{{rightarrow}^{hv}}mathrm{FAD}^{{bar{{cdot}}}}) end{document} photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.

Ultrafast solvation dynamics at binding and active sites of photolyases

Dynamic solvation at binding and active sites is critical to protein recognition and enzyme catalysis. We report here the complete characterization of ultrafast solvation dynamics at the recognition site of photoantenna molecule and at the active site of cofactor/substrate in enzyme photolyase by examining femtosecond-resolved fluorescence dynamics and the entire emission spectra. With direct use of intrinsic antenna and cofactor chromophores, we observed the local environment relaxation on the time scales from a few picoseconds to nearly a nanosecond. Unlike conventional solvation where the Stokes shift is apparent, we observed obvious spectral shape changes with the minor, small, and large spectral shifts in three function sites. These emission profile changes directly reflect the modulation of chromophore’s excited states by locally constrained protein and trapped-water collective motions. Such heterogeneous dynamics continuously tune local configurations to optimize photolyase’s function through resonance energy transfer from the antenna to the cofactor for energy efficiency and then electron transfer between the cofactor and the substrate for repair of damaged DNA. Such unusual solvation and synergetic dynamics should be general in function sites of proteins.

Electron tunneling pathways and role of adenine in repair of cyclobutane pyrimidine dimer by DNA photolyase

Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photoinitiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a superexchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds, while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways, and the critical role of adenine ensure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron-transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding that the forward and futile back-electron transfer is in the normal region and that the final electron return of the catalytic cycle is in the inverted region.

Purification and Characterization of a Type III Photolyase from Caulobacter crescentus

The photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases, and cryptochromes that regulate blue-light-dependent growth and development in plants, and light-dependent and light-independent circadian clock setting in animals. Phylogenetic analysis has revealed a new class of the family, named type III photolyase, which cosegregates with plant cryptochromes. Here we describe the isolation and characterization of a type III photolyase from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either both chromophores or only folate, we were able to determine the efficiency and rate of transfer of energy from MTHF to FAD.

Comparative photochemistry of animal type 1 and type 4 cryptochromes

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.

Femtochemistry in enzyme catalysis: DNA photolyase

Photolyase uses light energy to split UV-induced cyclobutane pyrimidine dimers in damaged DNA. This photoenzyme encompasses a series of elementary dynamical processes during repair function from early photoinitiation by a photoantenna molecule to enhance repair efficiency, to in vitro photoreduction through aromatic residues to reconvert the cofactor to the active form, and to final photorepair to fix damaged DNA. The corresponding series of dynamics include resonance energy transfer, intraprotein electron transfer, and intermolecular electron transfer, bond breaking-making rearrangements and back electron return, respectively. We review here our recent direct studies of these dynamical processes in real time, which showed that all these elementary reactions in the enzyme occur within subnanosecond timescale. Active-site solvation was observed to play a critical role in the continuous modulation of catalytic reactions. As a model system for enzyme catalysis, we isolated the enzyme–substrate complex in the transition-state region and mapped out the entire evolution of unmasked catalytic reactions of DNA repair. These observed synergistic motions in the active site reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.

Direct observation of DNA repair by photolyase

Author Institution: Department of Physics, Chemistry and Biochemistry, OSU Biophysics, Chemical Physics and Biochemistry Programs, The Ohio State University, Columbus, Ohio 43210