The nature of proton-coupled electron transfer in a blue light using flavin domain.

Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

Direct Nanosecond Multiframe Imaging of Irreversible Dynamics in 4D Electron Microscopy.

Irreversible ultrafast events are prevalent in nature, yet their capture in real time poses significant challenges. Traditional single-shot imaging technologies, which utilize a single optical pump and single delayed electron probe, offer high spatiotemporal resolution but fail to capture the entire dynamic evolutions. Here, we introduce a novel imaging method employing a single optical pump and delayed multiple electron probes. This approach, facilitated by an innovative deflector in ultrafast electron microscopy, enables the acquisition of nine frames per exposure, paving the way for statistical and quantitative analyses. We have developed an algorithm that corrects frame-by-frame distortions, realizing a cross-correlation enhancement of ∼26%. Achieving ∼12 nm and 20 ns resolution, our method allows for the comprehensive visualization of laser-induced behaviors in Au nanoparticles, including merging, jumping, and collision processes. Our results demonstrate the capability of this multiframe imaging technique to document irreversible processes across materials science and biology with unprecedented nanometer-nanosecond precision.

Dissecting the Ultrafast Stepwise Bidirectional Proton Relay in a Blue-Light Photoreceptor.

Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)-glutamic acid (E)-tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN·-/E-COOH/WH·+, the proton first rapidly jumps from the bridging E-COOH to FMN- (τfPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH·+ to E-COO- (τfPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W· (τrPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH· to E-COO- (τrPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN·- and WH·+ to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein.

An ultrafast phototrigger of the Trp5CN-Trp motif in a ß-hairpin peptide.

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN-W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm-1 from the CN stretch of the electron transfer intermediate W5CN·- in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W5CN-W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.

Revealing the origin of multiphasic dynamic behaviors in cyanobacteriochrome.

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.

Ultrafast Dynamics and Catalytic Mechanism of Fatty Acid Photodecarboxylase.

Fatty acid photodecarboxylase is a newly discovered flavin photoenzyme that converts a carboxylic acid into a hydrocarbon and a carbon dioxide molecule through decarboxylation. The enzymatic reactions are poorly understood. In this study, we carefully characterized its dynamic evolution with femtosecond spectroscopy. We observed initial electron transfer from the substrate to the flavin cofactor in 347 ps with a stretched dynamic behavior and subsequently captured the critical carbonyloxy radical. The dominant process following this step was decarboxylation in 5.8 ns to form an alkyl radical and a carbon dioxide molecule. We further identified the absorption bands of two carbonyloxy and alkyl radical intermediates. The overall enzymatic quantum efficiency determined by our obtained timescales is 0.81, consistent with the steady-state value. The results are essential to the elucidation of the enzyme mechanism and catalytic photocycle, providing a molecular basis for potential design of flavin-based artificial photoenzymes.

Direct Observation of Ultrafast Proton Rocking in the BLUF Domain.

We present direct observation of ultrafast proton rocking in the central motif of a BLUF domain protein scaffold. The mutant design has taken consideration of modulating the proton-coupled electron transfer (PCET) driving forces by replacing Tyr in the original motif with Trp, in order to remove the interference of a competing electron transfer pathway. Using femtosecond pump-probe spectroscopy and detailed kinetics analysis, we resolved an electron-transfer-coupled Grotthuss-type forward and reverse proton rocking along the FMN-Gln-Trp proton relay chain. The rates of forward and reverse proton transfer are determined to be very close, namely 51 ps vs. 52 ps. The kinetic isotope effect (KIE) constants associated with the forward and reverse proton transfer are 3.9 and 5.3, respectively. The observation of ultrafast proton rocking is not only a crucial step towards revealing the nature of proton relay in the BLUF domain, but also provides a new paradigm of proton transfer in proteins for theoretical investigations.

Ultrafast nonequilibrium dynamics of short-range protein electron transfer in flavodoxin.

Short-range protein electron transfer (ET) is ubiquitous in biology and is often observed in photosynthesis, photoreceptors and photoenzymes. These ET processes occur on an ultrafast timescale from femtoseconds to picoseconds at a short donor-acceptor distance within 10 Å, and thus couple with local environmental fluctuations. Here, we use oxidized Anabaena flavodoxin as a model system and have systematically studied the photoinduced redox cycle of the wild type and seven mutant proteins by femtosecond spectroscopy. We observed a series of ultrafast dynamics from the initial charge separation in 100-200 fs, subsequent charge recombination in 1-2 ps and final vibrational cooling process of the products in 3-6 ps. We further characterized the active-site solvation and observed the relaxations in 1-200 ps, indicating a nonergodic ET dynamics. With our new ET model, we uncovered a minor outer (solvent) reorganization energy and a large inner (donor and acceptor) reorganization energy, suggesting a frozen active site in the initial ultrafast ET while the back ET couples with the environment relaxations. The vibronically coupled back ET dynamics was first reported in D. vulgaris flavodoxin and here is observed in Anabaena flavodoxin again, completely due to the faster ET dynamics than the cooling relaxations. We also compared the two flavodoxin structures, revealing a stronger coupling with the donor tyrosine in Anabaena. All ultrafast ET dynamics are from the large donor-acceptor couplings and the minor activation barriers due to the reaction free energies being close to the inner reorganization energies. These observations should be general to many redox reactions in flavoproteins.

Exact eigenenergies of a model of vibronically coupled electron transfer reactions.

We present the first exact solution to the time-independent Schrödinger equation of a model Hamiltonian consisting of a vibrational mode coupled to three electronic states. This Hamiltonian serves as a generic model for photo-induced electronic transfer reactions. The solution is non-perturbative and can be applied to ET reactions with weak and strong electronic and vibrational coupling strengths. This work suggests a new direction towards understanding the vibronic effects in ET dynamics beyond the non-adiabatic limit and Condon approximation.

Ultrafast Dynamics of Water-Protein Coupled Motions around the Surface of Eye Crystallin.

Water dynamics on the protein surface mediate both protein structure and function. However, many questions remain about the role of the protein hydration layers in protein fluctuations and how the dynamics of these layers relate to specific protein properties. The fish eye lens protein γM7-crystallin (γM7) is found in vivo at extremely high concentrations nearing the packing limit, corresponding to only a few water layers between adjacent proteins. In this study, we conducted a site-specific probing of hydration water motions and side-chain dynamics at nine selected sites around the surface of γM7 using a tryptophan scan with femtosecond spectroscopy and NMR nuclear spin relaxation (NSR). We observed correlated fluctuations between hydration water and protein side chains on the time scales of a few picoseconds and hundreds of picoseconds, corresponding to local reorientations and network restructuring, respectively. These motions are heterogeneous over the protein surface and relate to the various steric and chemical properties of the local protein environment. Overall, we found that γM7 has relatively slower water dynamics within the hydration shell than a similar ß-sheet protein, which may contribute to the high packing limit of this unique protein.

Nonequilibrium dynamics of photoinduced forward and backward electron transfer reactions.

The biological functions of photoenzymes are often triggered by photoinduced electron transfer (ET) reactions. An ultrafast backward ET (BET) reaction follows the initial photoinduced forward ET (FET), which dissipates the energy of absorbed photons and terminates the biological function in vain. Based upon our previous works, we reasoned that the dynamics of the BET is coupled with that of the FET and other local motions. In this work, the dynamics of the FET and BET is modeled as the master equation of the reduced density operator of a three-state system coupled with a classical harmonic reservoir. The coupling of the FET and BET is reflected in the time-evolution of the charge-transfer state's population, which is generated by a source, the reaction flux for the FET, and annihilated by a sink, the reaction flux for the BET. Surprisingly, numerical simulations show that when the BET is in the Marcus normal region, the BET can be accelerated by nonequilibrium local motions and becomes faster than what is predicted by the Marcus theory. The experimental confirmation of this novel dynamics would provide qualitative evidence for nonequilibrium effects on ultrafast ET dynamics. Additionally, the effects of quantum vibrational modes on the dynamics are discussed. This work can help understand the dynamical interactions between the chain of ultrafast reactions and the complex local environmental motions, revealing the physical nature underlying biological functions.

Dynamics and mechanism of ultrafast water-protein interactions.

Protein hydration is essential to its structure, dynamics, and function, but water-protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship.

Trp triad-dependent rapid photoreduction is not required for the function of Arabidopsis CRY1.

Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photoreduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the "Trp triad." It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1(W324A) and CRY1(W400F) mutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.

Mapping Hydration Dynamics around a ß-Barrel Protein.

Protein surface hydration is fundamental to its structure, flexibility, dynamics, and function, but it has been challenging to disentangle their ultimate relationships. Here, we report our systematic characterization of hydration dynamics around a ß-barrel protein, rat liver fatty acid-binding protein (rLFABP), to reveal the effect of different protein secondary structures on hydration water. We employed a tryptophan scan to the protein surface one at a time and examined a total of 17 different sites. We observed three types of hydration water relaxation with distinct time scales, from hundreds of femtoseconds to a hundred picoseconds. We also examined the anisotropy dynamics of the corresponding tryptophan side chains and observed two distinct relaxations from tens to hundreds of picoseconds. Integrating our previous findings on α-helical proteins, we conclude the following: (1) The hydration dynamics is highly heterogeneous around the protein surface of both α-helical and ß-sheet proteins. The outer-layer water of the hydration shell behave like a bulk and relaxes in hundreds of femtoseconds. The inner-layer water collectively relaxes in two time scales, reorientation motions in a few picoseconds and network restructuring in tens to a hundred picoseconds. (2) The hydration dynamics are always faster than local protein relaxations and in fact drive the protein fluctuations on the picosecond time scale. (3) The hydration dynamics in general are more retarded around ß-sheet structures than α-helical motifs. A thicker hydration shell and a more rigid interfacial hydration network are observed in the ß-sheet protein. Overall, these findings elucidate the intimate relationship between water-protein interactions and dynamics on the ultrafast time scale.

Photolyase: Dynamics and electron-transfer mechanisms of DNA repair.

Photolyase, a flavoenzyme containing flavin adenine dinucleotide (FAD) molecule as a catalytic cofactor, repairs UV-induced DNA damage of cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) photoproduct using blue light. The FAD cofactor, conserved in the whole protein superfamily of photolyase/cryptochromes, adopts a unique folded configuration at the active site that plays a critical functional role in DNA repair. Here, we review our comprehensive characterization of the dynamics of flavin cofactor and its repair photocycles by different classes of photolyases on the most fundamental level. Using femtosecond spectroscopy and molecular biology, significant advances have recently been made to map out the entire dynamical evolution and determine actual timescales of all the catalytic processes in photolyases. The repair of CPD reveals seven electron-transfer (ET) reactions among ten elementary steps by a cyclic ET radical mechanism through bifurcating ET pathways, a direct tunneling route mediated by the intervening adenine and a two-step hopping path bridged by the intermediate adenine from the cofactor to damaged DNA, through the conserved folded flavin at the active site. The unified, bifurcated ET mechanism elucidates the molecular origin of various repair quantum yields of different photolyases from three life kingdoms. For 6-4 photoproduct repair, a similar cyclic ET mechanism operates and a new cyclic proton transfer with a conserved histidine residue at the active site of (6-4) photolyases is revealed.

Electron transfer mechanisms of DNA repair by photolyase.

Photolyase is a flavin photoenzyme that repairs two DNA base damage products induced by ultraviolet (UV) light: cyclobutane pyrimidine dimers and 6-4 photoproducts. With femtosecond spectroscopy and site-directed mutagenesis, investigators have recently made significant advances in our understanding of UV-damaged DNA repair, and the entire enzymatic dynamics can now be mapped out in real time. For dimer repair, six elementary steps have been characterized, including three electron transfer reactions and two bond-breaking processes, and their reaction times have been determined. A unique electron-tunneling pathway was identified, and the critical residues in modulating the repair function at the active site were determined. The dynamic synergy between the elementary reactions for maintaining high repair efficiency was elucidated, and the biological nature of the flavin active state was uncovered. For 6-4 photoproduct repair, a proton-coupled electron transfer repair mechanism has been revealed. The elucidation of electron transfer mechanisms and two repair photocycles is significant and provides a molecular basis for future practical applications, such as in rational drug design for curing skin cancer.

Dynamics and mechanism of repair of ultraviolet-induced (6-4) photoproduct by photolyase.

One of the detrimental effects of ultraviolet radiation on DNA is the formation of the (6-4) photoproduct, 6-4PP, between two adjacent pyrimidine rings. This lesion interferes with replication and transcription, and may result in mutation and cell death. In many organisms, a flavoenzyme called photolyase uses blue light energy to repair the 6-4PP (ref. 3). The molecular mechanism of the repair reaction is poorly understood. Here, we use ultrafast spectroscopy to show that the key step in the repair photocycle is acyclic proton transfer between the enzyme and the substrate. By femtosecond synchronization of the enzymatic dynamics with the repair function, we followed the function evolution and observed direct electron transfer from the excited flavin cofactor to the 6-4PP in 225 picoseconds, but surprisingly fast back electron transfer in 50 picoseconds without repair. We found that the catalytic proton transfer between a histidine residue in the active site and the 6-4PP, induced by the initial photoinduced electron transfer from the excited flavin cofactor to 6-4PP, occurs in 425 picoseconds and leads to 6-4PP repair in tens of nanoseconds. These key dynamics define the repair photocycle and explain the underlying molecular mechanism of the enzyme's modest efficiency.

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

The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.

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.

Characterization of the Intermediate in and Identification of the Repair Mechanism of (6-4) Photolesions by Photolyases.

Quantum mechanics/molecular mechanics calculations are employed to assign previously recorded experimental spectroscopic signatures of the intermediates occurring during the photo-induced repair of (6-4) photolesions by photolyases to specific molecular structures. Based on this close comparison of experiment and theory it is demonstrated that the acting repair mechanism involves proton transfer from the protonated His365 to the N3' nitrogen of the lesion, which proceeds simultaneously with intramolecular OH transfer along an oxetane-like transition state.