Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavine (vitamin B2)-binding protein and in glucose oxidase enzyme

Flavoproteins can function as hydrophobic sites for vitamin B(2) (riboflavin) or, in other structures, with cofactors for catalytic reactions such as glucose oxidation. In this contribution, we report direct observation of charge separation and recombination in two flavoproteins: riboflavin-binding protein and glucose oxidase. With femtosecond resolution, we observed the ultrafast electron transfer from tryptophan(s) to riboflavin in the riboflavin-binding protein, with two reaction times: approximately 100 fs (86% component) and 700 fs (14%). The charge recombination was observed to take place in 8 ps, as probed by the decay of the charge-separated state and the recovery of the ground state. The time scale for charge separation and recombination indicates the local structural tightness for the dynamics to occur that fast and with efficiency of more than 99%. In contrast, in glucose oxidase, electron transfer between flavin-adenine-dinucleotide and tryptophan(s)/tyrosine(s) takes much longer times, 1.8 ps (75%) and 10 ps (25%); the corresponding charge recombination occurs on two time scales, 30 ps and nanoseconds, and the efficiency is still more than 97%. The contrast in time scales for the two structurally different proteins (of the same family) correlates with the distinction in function: hydrophobic recognition of the vitamin in the former requires a tightly bound structure (ultrafast dynamics), and oxidation-reduction reactions in the latter prefer the formation of a charge-separated state that lives long enough for chemistry to occur efficiently. Finally, we also studied the influence on the dynamics of protein conformations at different ionic strengths and denaturant concentrations and observed the sharp collapse of the hydrophobic cleft and, in contrast, the gradual change of glucose oxidase.     

Effect of protein dynamics on biological electron transfer

Computer simulations of the effect of protein dynamics on the long distance tunneling mediated by the protein matrix have been carried out for a Ru-modified (His 126) azurin molecule. We find that the tunneling matrix element is a sensitive function of the atomic configuration of the part of the protein matrix in which tunneling currents (pathways) are localized. Molecular dynamics simulations show that fluctuations of the matrix element can occur on a time scale as short as 10 fs. These short time fluctuations are an indication of a strong dynamic coupling of a tunneling electron to vibrational motions of the protein nuclear coordinates. The latter results in a modification of the conventional Marcus picture of electron transfer in proteins. The new element in the modified theory is that the tunneling electron is capable of emitting or absorbing vibrational energy (phonons) from the medium. As a result, some biological reactions may occur in an activationless fashion. An analytical theoretical model is proposed to account for thermal fluctuations of the medium in long distance electron transfer reactions. The model shows that, at long distances, the phonon-modified inelastic tunneling always dominates over the conventional elastic tunneling.     

Charge conductivity in peptides: dynamic simulations of a bifunctional model supporting experimental data

Our previous finding and the given mechanism of charge and electron transfer in polypeptides are here integrated in a bifunctional model involving electronic charge transfer coupled to special internal rotations. Present molecular dynamics simulations that describe these motions in the chain result in the mean first passage times for the hopping process of an individual step. This "rest and fire" mechanism is formulated in detail-i.e., individual amino acids are weakly coupled and must first undergo alignment to reach the special strong coupling. This bifunctional model contains the essential features demanded by our prior experiments. The molecular dynamics results yield a mean first passage time distribution peaked at about 140 fs, in close agreement with our direct femtosecond measurements. In logic gate language this is a strongly conducting ON state resulting from small firing energies, the system otherwise being a quiescent OFF state. The observed time scale of about 200 fs provides confirmation of our simulations of transport, a model of extreme transduction efficiency. It explains the high efficiency of charge transport observed in polypeptides. We contend that the moderate speed of weak coupling is required in our model by the bifunctionality of peptides. This bifunctional mechanism agrees with our data and contains valuable features for a general model of long-range conductivity, final reactivity, and binding at a long distance.     

Ultrafast detection and control of molecular dynamics

Many elementary chemical and physical processes such as the breaking of a chemical bond or the vibrational motion of atoms within a molecule take place on a femtosecond (fs = 10(-15)s) or picosecond (ps = 10(-12)s) time scale. It is now possible to monitor these events as a function of time with temporal resolution well below 100 fs. This capability is based on the pump-probe technique where one optical pulse triggers a reaction and a second delayed optical pulse probes the changes that ensue. To illustrate this capability, the dynamics of ligand motion within a protein are presented. Moving beyond casual observation of a reaction to active control of its outcome requires additional experimental and theoretical effort. To illustrate the concept of control, the effect of optical pulse duration on the vibrational dynamics of a tri-atomic molecule are discussed. The experimental and theoretical resources currently available are poised to make the dream of reaction control a reality for certain molecular systems.     

Complementarity of ensemble and single-molecule measures of protein motion: a relaxation dispersion NMR study of an enzyme complex

Single-molecule fluorescence experiments have shown that the conformation of the complex between Escherichia coli general NAD(P)H:flavin oxidoreductase (FRE) and flavin adenine dinucleotide (FAD) fluctuates over a range of timescales between 10(-4) and 1 s. Here we use (15)N and (13)C relaxation dispersion NMR methods to study millisecond-timescale dynamics in the complex. In this time regime, the protein is extremely flexible, with residues that undergo conformational exchange located throughout the molecule. Three distinct regions of dynamics are quantified, with two of them involving residues making contact to the donor (Tyr-35) and acceptor (FAD) sites that participate in the electron transfer reaction monitored in single-molecule experiments. Modulation of the donor-acceptor distance through these conformational exchange processes, occurring with rates of approximately 400 and 1,200 s(-1) (22 degrees C), affects the rate of electron transfer and partially accounts for the range of the observed dynamics monitored in the fluorescence experiments.     

Chromophore-protein interactions and the function of the photosynthetic reaction center: a molecular dynamics study.

The coupling between electron transfer and protein structure and dynamics in the photosynthetic reaction center of Rhodopseudomonas viridis is investigated. For this purpose molecular dynamics simulations of the essential portions (a segment of 5797 atoms) of this protein complex have been carried out. Electron transfer in the primary event is modeled by altering the charge distributions of the chromophores according to quantum chemical calculations. The simulations show (i) that fluctuations of the protein matrix, which are coupled electrostatically to electron transfer, play an important role in controlling the electron transfer rates and (ii) that the protein matrix stabilizes the separated electron pair state through rapid (200 fs) and temperature-independent dielectric relaxation. The photosynthetic reaction center resembles a polar liquid in that the internal motions of the whole protein complex, rather than only those of specific side groups, contribute to i and ii. The solvent reorganization energy is about 4.5 kcal/mol. The simulations indicate that rather small structural rearrangements and changes in motional amplitudes accompany the primary electron transfer.     

A quantitative structure-function relationship for the Photosystem II reaction center: supermolecular behavior in natural photosynthesis

Light-induced charge separation is the primary photochemical event of photosynthesis. Efficient charge separation in photosynthetic reaction centers requires the balancing of electron and excitation energy transfer processes, and in Photosystem II (PSII), these processes are particularly closely entangled. Calculations that treat the cofactors of the PSII reaction center as a supermolecular complex allow energy and electron transfer reactions to be described in a unified way. This calculational approach is shown to be in good agreement with experimentally observed energy and electron transfer dynamics. This supermolecular view also correctly predicts the effect of changing the redox potentials of cofactors by site-directed mutagenesis, thus providing a unified and quantitative structure-function relationship for the PSII reaction center.     

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

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.     

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (tau(D) congruent with 200 micros, "peroxy" to "ferryl" transition) followed by a significantly slower proton release to the other side of the membrane (tau(D) congruent with 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.     

Visualizing electron rearrangement in space and time during the transition from a molecule to atoms

Imaging and controlling reactions in molecules and materials at the level of electrons is a grand challenge in science, relevant to our understanding of charge transfer processes in chemistry, physics, and biology, as well as material dynamics. Direct access to the dynamic electron density as electrons are shared or transferred between atoms in a chemical bond would greatly improve our understanding of molecular bonding and structure. Using reaction microscope techniques, we show that we can capture how the entire valence shell electron density in a molecule rearranges, from molecular-like to atomic-like, as a bond breaks. An intense ultrashort laser pulse is used to ionize a bromine molecule at different times during dissociation, and we measure the total ionization signal and the angular distribution of the ionization yield. Using this technique, we can observe density changes over a surprisingly long time and distance, allowing us to see that the electrons do not localize onto the individual Br atoms until the fragments are far apart (∼5.5 Å), in a region where the potential energy curves for the dissociation are nearly degenerate. Our observations agree well with calculations of the strong-field ionization rates of the bromine molecule.     

Dissection of complex protein dynamics in human thioredoxin

We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47-0.67 and 10.8-13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10-17.5 ps for three mutants. A robust dynamical process in 95-114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275-615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.     

Direct observation of thymine dimer repair in DNA by photolyase

Photolyase uses light energy to split UV-induced cyclobutane dimers in damaged DNA, but its molecular mechanism has never been directly revealed. Here, we report the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We observed direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site, with no net change in the redox state of the flavin cofactor.     

Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.

Protein and cofactor vibrational dynamics associated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femto-second (300-400 fs) time-resolved infrared (1560-1960 cm-1) spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation modifies the polypeptide carbonyl spectra. The potential energy surfaces of the "special pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process. Kinetic signatures of all other transient intermediates, P, P*, and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.     

Probing conformational dynamics in single donor-acceptor synthetic molecules by means of photoinduced reversible electron transfer

We use single-molecule fluorescence lifetimes to probe dynamics of photoinduced reversible electron transfer occurring between triphenylamine (donor) and perylenediimide (acceptor) in single molecules of a polyphenylenic rigid dendrimer embedded in polystyrene. Here, reversible electron transfer in individual donor-acceptor molecules results in delayed fluorescence that is emitted with a high photon count rate. By monitoring fluorescence decay times on a photon-by-photon basis, we find fluctuations in both forward and reverse electron transfer spanning a broad time range, from milliseconds to seconds. Fluctuations are induced by conformational changes in the dendrimer structure as well by polystyrene chain reorientation. The conformational changes are related to changes in the dihedral angle of adjacent phenyl rings located in the dendritic branch near the donor transferring the charge, a torsional motion that results in millisecond fluctuations in the "through-bond" donor-acceptor electronic coupling. Polymer chain reorientation leads to changes in the local polarity experienced by the donors and to changes in the solvation of the charge-separated state. As a result, switching between different donor moieties within the same single molecule becomes possible and induces fluctuations in decay time on a time scale of seconds.     

Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer.

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding "molecular excited states." Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(-)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.     

The first step in vision occurs in femtoseconds: complete blue and red spectral studies.

Femtosecond transient absorption measurements of the cis-trans isomerization of the visual pigment rhodopsin clarify the interpretation of the dynamics of the first step in vision. We present femtosecond time-resolved spectra as well as kinetic measurements at specific wavelengths between 490 and 670 nm using 10-fs probe pulses centered at 500 and 620 nm following a 35-fs pump pulse at 500 nm. The expanded spectral window beyond that available (500-570 nm) in our previous study [Schoenlein, R. W., Peteanu, L. A., Mathies, R. A. & Shank, C. V. (1991) Science 254, 412-415] provides the full differential absorption spectrum of the photoproduct as a function of delay time after photolysis. The high time-resolution data presented here contradict an alternative interpretation of the rhodopsin photochemistry offered by Callender and co-workers [Yan, M., Manor, D., Weng, G., Chao, H., Rothberg, L., Jedju, T. M., Alfano, R. R. & Callender, R. H. (1991) Proc. Natl. Acad. Sci. USA 88, 9809-9812]. Our results confirm that the red-shifted (lambda max approximately 570 nm) photo-product of the isomerization reaction is fully formed within 200 fs. Subsequent changes in the differential spectra between 200 fs and 6 ps are attributed to a combination of dynamic ground-state processes such as intramolecular vibrational energy redistribution, vibrational cooling, and conformational relaxation.     

Femtosecond observation of benzyne intermediates in a molecular beam: Bergman rearrangement in the isolated molecule

In this communication, we report our femtosecond real-time observation of the dynamics for the three didehydrobenzene molecules (p-, m-, and o-benzyne) generated from 1,4-, 1,3-, and 1, 2-dibromobenzene, respectively, in a molecular beam, by using femtosecond time-resolved mass spectrometry. The time required for the first and the second C-Br bond breakage is less than 100 fs; the benzyne molecules are produced within 100 fs and then decay with a lifetime of 400 ps or more. Density functional theory and high-level ab initio calculations are also reported herein to elucidate the energetics along the reaction path. We discuss the dynamics and possible reaction mechanisms for the disappearance of benzyne intermediates. Our effort focuses on the isolated molecule dynamics of the three isomers on the femtosecond time scale.     

NOD2/CARD15基因R702W、G908R及L1007fs多态性与广西壮族人群炎症性肠病的相关性

目的:探讨我国广西壮族人群NOD2/CARD15基因R702W、G908R及L1007fs的遗传多态性与炎症性肠病的相关性.方法:分别收集2007-02/2010-10在广西地区无亲缘关系的壮族(n=70)和汉族(n=76)IBD患者及壮族(n=80)和汉族(n=84)正常对照者的肠黏膜组织.采用酚氯仿法提取各组织样本DNA,采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)方法对NOD2/CARD15基因R702W、G908R及L1007fs进行检测,统计基因型及等位基因频率,分析上述3个多态性位点与广西壮族人群炎症性肠病的相关性.结果:广西壮族和汉族IBD患者与正常对照者均未发现NOD2/CARD15基因R702W、G908R及L1007fs突变型基因型,所有多态性位点上的基因型全部为野生型纯合子,其基因型频率和等位基因频率分布在IBD患者和正常对照者中差异无统计学意义(P>0.05).结论:NOD2/CARD15基因R702W、G908R及L1007fs多态性与广西壮族人群炎症性肠病无明显相关性.     

Femtosecond direct observation of charge transfer between bases in DNA

Charge transfer in supramolecular assemblies of DNA is unique because of the notion that the pi-stacked bases within the duplex may mediate the transport, possibly leading to damage and/or repair. The phenomenon of transport through pi-stacked arrays over a long distance has an analogy to conduction in molecular electronics, but the mechanism still needs to be determined. To decipher the elementary steps and the mechanism, one has to directly measure the dynamics in real time and in suitably designed, structurally well characterized DNA assemblies. Here, we report our first observation of the femtosecond dynamics of charge transport processes occurring between bases within duplex DNA. By monitoring the population of an initially excited 2-aminopurine, an isomer of adenine, we can follow the charge transfer process and measure its rate. We then study the effect of different bases next to the donor (acceptor), the base sequence, and the distance dependence between the donor and acceptor. We find that the charge injection to a nearest neighbor base is crucial and the time scale is vastly different: 10 ps for guanine and up to 512 ps for inosine. Depending on the base sequence the transfer can be slowed down or inhibited, and the distance dependence is dramatic over the range of 14 A. These observations provide the time scale, and the range and efficiency of the transfer. The results suggest the invalidity of an efficient wire-type behavior and indicate that long-range transport is a slow process of a different mechanism.