Ultrafast solvation dynamics at binding and active sites of photolyases

Proc. Natl. Acad. Sci. U.S.A.

Guo

et al. 2011

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144

250

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.DNA repair photocycle | ultrafast enzyme dynamics | thymine dimer splitting | electron tunneling pathway | active-site mutation U ltraviolet (UV) component of sunlight irradiation causes DNA damage by inducing the formation of cyclobutane pyrimidine dimer (CPD), which is mutagenic and a leading cause of skin cancer (1-3). CPD can be completely restored by a photoenzyme, photolyase, through absorption of visible blue light (4). In our early work (5-7), we have observed a cyclic electron-transfer (ET) reaction in thymine dimer (ThiT) repair by photolyase and determined the time scale of 700 ps for the complete repair photocycle (7). However, the central questions of whether the splitting of the cyclobutane ring is synchronously or asynchronously concerted or stepwise and whether the cyclic ET involves specific tunneling pathways were not resolved. Furthermore, the molecular mechanism underlying the high repair efficiency has not been elucidated. Here, using femtosecond spectroscopy and site-directed mutagenesis, we are able to measure the dynamics of all initial reactants, reaction intermediates, and final products with different substrates and with wild-type and active-site mutant enzymes, and thus reveal the complete spatio-temporal molecular picture of thymine dimer repair by photolyase.Photolyase contains a fully reduced flavin adenine dinucleotide (FADH − ) as the catalytic cofactor and electron donor (4). Based on previous studies (4-10), a sequential repair mechanism of thymine dimer splitting is shown in Fig. 1. Previously, we found that the forward ET from FADH − Ã to ThiT occurs in 250 ps (1∕k FET ) and the total decay of intermediate FADH• in 700 ps (1∕k total ) (5, 7). These dynamics usually follow a stretched-exponential decay behavior, reflecting heterogeneous ET dynamics controlled by the active-site solvation (5, 6, 11). However, in that study, no thymine-related species could be detected in the vi...

mentioning

confidence: 54%

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Liu

Proc. Natl. Acad. Sci. U.S.A.

Guo

et al. 2011

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144

250

“…2a and Supplementary Fig. 1a), Ae À ðt=tÞ b (A, amplitude; t, decay time constant; and b, stretched parameter), due to the modulation of ET by active-site solvation on similar timescales [11][12][13]19,20 . Using t h i ¼ ðt=bÞGð1=bÞ and knowing the deactivation lifetimes (t LT ) in nanoseconds in the absence of substrate (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

The molecular origin of high DNA-repair efficiency by photolyase

Liu

et al. 2015

Nat Commun

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The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptor are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency. Here, we analyse the primary reaction steps of repair of ultraviolet-damaged DNA by photolyase using femtosecond spectroscopy. With systematic mutations of the amino acids involved in binding of the flavin cofactor and the cyclobutane pyrimidine dimer substrate, we report our direct deconvolution of the catalytic dynamics with three electron-transfer and two bond-breaking elementary steps and thus the fine tuning of the biological repair function for optimal efficiency. We found that the maximum repair efficiency is not enhanced by the ultrafast photoinduced process but achieved by the synergistic optimization of all steps in the complex repair reaction.

“…We have to emphasize that the ET reactions are coupled with active-site relaxation. We have measured the active-site solvation dynamics in the reduced FADH -state and have found that the relaxation takes place on the timescales from a few picoseconds to subnanoseconds (21). The similar timescales would be expected for the oxidized FAD state (22) and therefore the ET dynamics shows a stretched behavior, Ae −ðt=τÞ β , where A is the amplitude, τ is the lifetime, and β here is a stretched parameter.…”

Section: Identifying New Electron Donors (Ade and W384) In Initial Elmentioning

confidence: 96%

“…The derived ET values of free energies and reorganization energies from Eq. 1 are under assumption of equilibrium ET processes, and clearly these ET dynamics reported here are in nonequilibrium with local protein and solvent relaxations due to the overlap of timescales between ET dynamics and active-site relaxation (21). Woiczikowski et al (32) recently reported nonadiabatic quantum mechanics/molecular mechanics simulations of these ET dynamics and also showed the nonequilibrium nature of these tunneling processes.…”

Section: Characterizing Terminal Electron Donor (W306) and Completingmentioning

confidence: 99%

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Liu¹,

Proc. Natl. Acad. Sci. U.S.A.

Guo³

et al. 2013

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170

The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution. With various site-directed mutations, we identified all possible electron donors in the enzyme and determined their ET timescales. The excited cofactor behaves as an electron sink to draw electron flow from a series of encircling aromatic molecules in three distinct layers from the active site in the center to the protein surface. The dominant electron flow follows the conserved tryptophan triad in a hopping pathway across the layers with multiple tunneling steps. These ET dynamics occur ultrafast in less than 150 ps and are strongly coupled with local protein and solvent relaxations. The reverse electron flow from the flavin is slow and in the nanosecond range to ensure high reduction efficiency. With 12 experimentally determined elementary ET steps and 6 ET reaction pairs, the enzyme exhibits a distinct reduction-potential gradient along the same aromatic residues with favorable reorganization energies to drive a highly unidirectional electron flow toward the active-site center from the protein surface.protein electron transfer | flavin photoreduction | femtosecond dynamics | electron flow directionality | reduction potential funnel P hotolyase and cryptochrome are evolutionally related and contain a flavin adenine dinucleotide (FAD) as the catalytic cofactor with a unique bent structure in the active sites, but the two perform different functions: photolyase repairs UV-damaged DNA and cryptochrome functions as a photoreceptor for regulation of plant growth or synchronization of circadian rhythm (1-5). The active state of the cofactor in vivo is in the anionic hydroquinone form (FADH -) in photolyase (6), but currently the redox status of flavin in cryptochrome is under debate with some studies suggesting flavin to be in oxidized (FAD), whereas others claiming anionic (FAD -/FADH -) states for the functional form in vivo (7-10). However, in vitro, the cofactor is oxidized to FAD and/or FADH • in photolyase but only appears in the oxidized FAD state in cryptochrome. Thus, it appears that reduction of the oxidized FAD is necessary for its transformation into the active state. Photoinduced electron transfer (ET) is an effective way for such redox state changes and it is conceivable that the photoreduction of FAD could be a primary process for signal initiation in cryptochrome (11).In this study, we used Escherichia coli photolyase (EcPL) as a model system for systematic studies of electron flow into the excited cofactor FAD. As shown in Fig. 1, more than 10 aromatic acid residues (W and Y) encircle the flavin cofactor around the active site (12). These aromatic residues not only are cri...