We report here our systematic studies of excited-state dynamics of two common flavin molecules, FMN and FAD, in five redox states of 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 are 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 all 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 in nanoseconds except for the stacked conformation of FAD, where the intramolecular electron transfer between the ring and the adenine moiety in 5-9 ps as well as the subsequent charge recombination in 30-40 ps were observed. These observed distinct dynamics, controlled by the flavin ring flexibility, are fundamental to flavoenzyme's 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.
Optimizing the ratio of the rates for charge separation (CS) over charge recombination (CR) is crucial to create long-lived charge-separated states. Mastering the factors that govern the electron transfer (ET) rates is essential when trying to achieve molecular-scale electronics, artificial photosynthesis, and also for the further development of solar cells. Much work has been put into the question of how the donor-acceptor distances and donor-bridge energy gaps affect the electronic coupling, V(DA), and thus the rates of ET. We present here a unique comparison on how these factors differently influence the rates for CS and CR in a porphyrin-based donor-bridge-acceptor model system. Our system contains three series, each of which focuses on a separate charge-transfer rate-determining factor, the donor-acceptor distance, the donor-bridge energy gap, and last, the influence of the electron acceptor on the rate for charge transfer. In these three series both CS and CR are governed by superexchange interactions which make a CR/CS comparative study ideal. We show here that the exponential distance dependence increases slightly for CR compared to that for CS as a result of the increased tunneling barrier height for this reaction, in accordance with the McConnell superexchange model. We also show that the dependence on the tunneling barrier height is different for CS and CR. This difference is highly dependent on the electron acceptor and thus cannot solely be explained by the differences in the frontier orbitals of the electron donor in these porphyrin systems.
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 femtosecondresolved 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.function-site solvation | ultrafast dynamics | spectral tuning | protein rigidity and flexibility | femtosecond-resolved emission spectra D ynamic solvation in binding and active sites plays a critical role in protein recognition and enzyme reaction, and such local motions optimize spatial configurations and minimize energetic pathways (1-11). These dynamics involve local constrained protein and trapped-water motions within angstrom distance and occur on ultrafast time scales (6,7,10,11). Typically, extrinsic dye molecules or synthetic amino acids were used as local optical probes to label function sites, and the local relaxations were observed, ranging from femtoseconds to nanoseconds (5,(12)(13)(14). Such labeling of bulky dye molecules usually induces significant local perturbations, and direct characterization with intrinsic chromophores in proteins eliminates those interferences and reveals intact environment responses (4, 7, 15-21), as recently examined in green fluorescence proteins (21). We have recently studied a series of flavoproteins using intrinsic flavin molecule as the optical probe (10, 22, 23) and especially found the important functional role of local solvation in photolyase (10).Photolyase, a flavoprotein and a photoenzyme, repairs damaged DNA caused by UV irradiation. Two types of structurally similar photolyases are specific for two major UV-induced DNA lesions of cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (24). The CPD photolyase contains two noncovalently bound chromophores, a pterin molecule in the form of methenyltetrahydrofolate (MTHF) in the binding site as the photoantenna for energy efficiency and a fully reduced flavin adenine dinucleotide (FADH − ) at the active site as the catalytic cofactor for repair of d...
Flavoproteins are unique redox coenzymes and the dynamic solvation at their function sites is critical to the understanding of their electron-transfer properties. Here, we report our complete characterization of the function-site solvation of holoflavodoxin in three redox states and of the binding-site solvation of apoflavodoxin. Using intrinsic flavin cofactor and tryptophan residue as the local optical probes with two site-specific mutations, we observed distinct ultrafast solvation dynamics at the function site in the three states and at the related recognition site of the cofactor, ranging from a few to hundreds of picoseconds. The initial ultrafast motion in 1-2.6 ps reflects the local water-network relaxation around the shallow, solvent-exposed function site. The second relaxation in 20-40 ps results from the coupled local water-protein fluctuation. The third dynamics in hundreds of picoseconds is from the intrinsic fluctuation of the loose loops flanking the cofactor at the function site. These solvation dynamics with different amplitudes well correlate with the redox states from the oxidized form, to the more rigid semiquinone and to the much looser hydroquinone. This observation of the redox control of local protein conformation plasticity and water network flexibility is significant and such an intimate relationship is essential to the biological function of interprotein electron transfer.
We report here our systematic studies of the heme dynamics and induced protein conformational relaxations in two redox states of ferric and ferrous cytochrome c upon femtosecond excitation. With a wide range of probing wavelengths from the visible to the UV and a site-directed mutation we unambiguously determined that the protein dynamics in the two states are drastically different. For the ferrous state the heme transforms from 6-fold to 5-fold coordination with ultrafast ligand dissociation in less than 100 fs, followed by vibrational cooling within several picoseconds, but then recombining back to its original 6-fold coordination in 7 ps. Such impulsive bond breaking and late rebinding generate proteinquakes and strongly perturb the local heme site and shake global protein conformation, which were found to completely recover in 13 and 42 ps, respectively. For the ferric state the heme however maintains its 6-fold coordination. The dynamics mainly occur at the local site, including ultrafast internal conversion in hundreds of femtoseconds, vibrational cooling on the similar picosecond time scale, and complete ground-state recovery in 10 ps, and no global conformation relaxation was observed.
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