Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Abstract

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...

Dynamic determination of the functional state in photolyase and the implication for cryptochrome

“…Tuning the probe wavelengths to shorter than 700 nm to search for the maximum ) and leads to the completion of the redox cycle. As discussed in the preceding paper (16), such ET dynamics between the Lf and Ade moieties is favorable by negative free-energy changes.…”

The molecular origin of high DNA-repair efficiency by photolyase

“…For all the mutants, the FET dynamics with the exception of M345A become slower and all the BET processes become faster, and thus unfavourable for repair. To understand these changes, we evaluated these ET reactions using the Marcus ET theory to estimate the driving forces ( À DG 0 ) and reorganization energies (l) [23][24][25][26] for each form of the enzyme (see Supplementary Note 2). Figure 3c shows the derived results of DG 0 and l for the FET, BET and ER.…”

“…The ER after repair is in the Marcus inverted region ( À DG 0 Zl). The mutations considerably alter not only the free energy changes, that is, the reduction potentials of the cofactor or the substrate, but also the reorganization energies 20,25,26 and thus significantly modulate all three ET reactions. The derived large reorganization energies mainly come from the contributions of the different structural distortions of FADH À and FADH (refs 25,27).…”

Direct observation of light-driven, concerted electron–proton transfer

“…An important, if largely unrecognized, role for photoEPT could exist and play a role broadly, for example, in DNA photodamage (21,22) or in forming reactive oxygen intermediates (ROS) (23). Nonetheless, reports of photoEPT and its role in excited state reactivity in chemistry and biology are rare (17-20, 24, 25).…”