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.     

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH^-). The CPD repair mechanism involves electron transfer from photoexcited FADH^- to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH^-. The two electron transfer processes occur on time scales of 10^-10 and 10^-9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T^∘-) and electron return from T^∘- to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.     

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease–related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme b_H of cytochrome bc₁, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme b_H caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Hemes are common redox-active cofactors in biological electron transfer systems. Their major function is to transfer electrons within the cofactor chains or as part of the catalytic sites. The direction and rate of electron transfer are secured by the specific properties of hemes, among which the redox midpoint potential and the spin state are considered to be of crucial importance. Several studies have been carried out to understand how specific molecular elements contribute in adjusting the redox potential values to the levels required for efficient electron transfer rate (1–5). These studies recognized the importance of the heme-iron ligands, the degree of exposure of the heme to the solvent, and specific interactions, including hydrogen bonding, within the amino acids of heme-binding pocket. However, the overall structural complexity of proteins often makes an experimental extraction of individual elements of control extremely challenging, in particular those associated within the hydrogen bonding networks. It is indeed remarkable that the fluctuation of the hydrogen bond network was identified as the factor modulating the efficiency of long-range biological electron transfer (6, 7).This work focused on exploring the hydrogen bond-related elements of control of the redox properties of one of the hemes of cytochrome b subunit of cytochrome bc₁ (mitochondrial complex III). Cytochrome b is the only subunit of this complex encoded by mitochondrial DNA, thus is a subject of high susceptibility for spontaneous mutations in comparison to other subunits (encoded by nuclear DNA). Such mutations often lead to systemic disorders, mitochondrial diseases, manifesting in symptoms such as exercise intolerance, myopathies, or neuropathies. On the other hand, their mimic in the bacterial or yeast model systems not only provides insights into the possible molecular basis of the disease but also reveal molecular aspects of protein design (8, 9). With this in mind, we targeted a mutation G34S (human cytochrome b numbering), found in one 52-y-old female patient suffering from exercise intolerance (10). The mutation was present in mitochondria from muscle tissue and caused mild myopathy, lactic acidosis, and defect in mitochondrial complex III activity. The mutated glycine 34 is located on transmembrane helix A of cytochrome b subunit in close distance (∼8 Å) to heme b_H (SI Appendix, Fig. S1 A and B). This location suggests the possible effect of mutation on the properties of heme cofactor, in particular if one considers that a change of small and nonpolar glycine to a larger and polar serine may affect the local hydrogen bonding environment.Indeed, the presence of glycine at position 34 was previously noticed as important for heme packing in the cytochrome b subunit (11). Interestingly, the importance of G34 was emphasized by the remarkable evolutionary conservation in the equivalents of cytochrome bc₁ complex in distant organisms, such as bacteria, insects, fish, mammals and even plants (12). In a bacterial model (Rhodobacter sphaeroides cytochrome bc₁), the equivalent G48 residue was mutated to valine and aspartic acid, both rendering the bacteria photosynthetically incompetent (13). The equivalent of this mutation was also studied in Saccharomyces cerevisiae (8). Yeast with G33S (S. cerevisiae numbering) mutation were not able to grow aerobically, whereas the isolated bc₁ complex had lower enzymatic activity and disrupted subunit composition (lower level of the iron-sulfur protein assessed by Western blotting and lower cytochrome b content, measured optically to 55% of wild type [WT]). The same G33 position was found to be mutated to aspartic acid in a respiratory-deficient yeast (14). Among the 85 tested G33D revertants, 82 were D33G and 3 were D33A, suggesting that only a small amino acid without a charge can be tolerated at this position.Given the implicated importance of glycine at 34 position of mitochondrial cytochrome b, particularly in the context of hydrogen bonding network within the heme-binding pocket, we combined the experimental and computational methods to investigate the effects of introducing serine to the homologous position in purple photosynthetic bacterium Rhodobacter capsulatus (mutant G48S). Notably, the bacterial cytochrome b shares reasonable sequence similarity with human and yeast mitochondrial cytochrome b, close to 57%. We found that G48S perturbs the redox properties of heme b_H and causes structural and hydration changes in the vicinity of heme.The mutation also severely affects the spectral properties of heme b_H, which are best explained by a model assuming a reversible change from low to high spin state when the heme is oxidized. The latter comes as a rather unusual and unexpected molecular effect, considering all so-far reported effects of disease-related mitochondrial mutations (8, 9, 15). At the same time, it offers interesting insights into the role of hydrogen bonding and protein packing in maintaining the low spin state of the oxidized heme fostering electron-transfer relay function.     

Ultrafast X-ray scattering offers a structural view of excited-state charge transfer

Intramolecular charge transfer and the associated changes in molecular structure in N,N′-dimethylpiperazine are tracked using femtosecond gas-phase X-ray scattering. The molecules are optically excited to the 3p state at 200 nm. Following rapid relaxation to the 3s state, distinct charge-localized and charge-delocalized species related by charge transfer are observed. The experiment determines the molecular structure of the two species, with the redistribution of electron density accounted for by a scattering correction factor. The initially dominant charge-localized state has a weakened carbon–carbon bond and reorients one methyl group compared with the ground state. Subsequent charge transfer to the charge-delocalized state elongates the carbon–carbon bond further, creating an extended 1.634 Å bond, and also reorients the second methyl group. At the same time, the bond lengths between the nitrogen and the ring-carbon atoms contract from an average of 1.505 to 1.465 Å. The experiment determines the overall charge transfer time constant for approaching the equilibrium between charge-localized and charge-delocalized species to 3.0 ps.

Understanding the dynamic process of photoinduced charge transfer is expected to lead to many practical applications, including efficient photovoltaic systems, the development of photocatalysts, and better materials for energy storage (1–3). Charge transfer redistributes the electrons in a molecule and is typically associated with changes in the molecular geometry (2). On the fastest time scale, electrons move so rapidly that the nuclei appear frozen, a phenomenon known as charge migration (4–10). When time scales approach the typical vibrational motions of molecules, that is, tens of femtoseconds (10⁻¹⁴ s), the nuclei can adjust their positions, often resulting in localization of electronic charge and permanent changes in molecular geometry (11). While exhibiting a rich phenomenology on different time scales, it is evident that electron charge transfer and nuclear dynamics are intrinsically coupled (12–17). An accurate determination of the changes in molecular structure during charge transfer is therefore of great interest from both applied and fundamental perspectives.New scientific technologies, in particular X-ray free-electron lasers (XFEL) (18, 19) and MeV ultrafast electron diffraction (20), have made it possible to study structural dynamics in the ultrafast regime. Recent femtosecond gas-phase scattering experiments have successfully tracked structural changes during chemical reactions (21–26) and probed specific signatures of excited electronic states (27, 28). Given the emerging ability of ultrafast gas-phase scattering to record both nuclear and electronic structure (25, 27, 28), we use time-resolved gas-phase X-ray scattering to study the photoinduced intramolecular charge transfer in an organic molecule, N,N′-dimethylpiperazine (DMP, C₆H₁₄N₂), shown in Fig. 1. In its ground electronic state, DMP has C_2h symmetry with two equivalent ionization centers, one on each nitrogen atom. Valence ionization, or in the present case excitation to an electronic Rydberg state (29), induces charge transfer between the two nitrogen atoms, making DMP a prototype for exploring electron lone-pair interactions and charge transfer (30–32).Open in a separate windowFig. 1.A schematic illustration of the experimental setup. The ground-state molecules (DMP) were excited by 200 nm ultraviolet pump pulses, and the transient structures were probed by 9.5 keV X-ray pulses at variable time delays. The scattering signals were recorded on a CSPAD detector. The inset shows the calculated spin density, which gives the difference in density of electrons with spin up and spin down, of the charge-localized DMP (3sL) and charge-delocalized DMP (3sD) in the 3s Rydberg states at isovalues of 0.1 electron/ų.Previously, energy relaxation pathways and charge transfer in electronically excited DMP were explored using Rydberg fingerprint spectroscopy (33), a form of photoelectron spectroscopy. As depicted in Scheme 1, the investigations by Deb et al. (33) found that optical excitation at 207 nm prepares the molecule in a 3p Rydberg state, creating a state with a localized charge in the molecular core (3pL). Internal conversion to 3s then leads to charge-localized (3sL) and charge-delocalized (3sD) conformers with 230 and 480 fs time constants, respectively. The charge transfer proceeds as the molecules explore the 3s potential energy surface. An equilibrium between 3sL and 3sD structures is eventually established with an overall time constant of 2.65 ps, with the forward and backward first-order kinetic time constants for the transformation 3.4 and 12.0 ps, respectively (33).Open in a separate windowScheme 1.Reaction pathway for Rydberg-excited DMP as determined previously (33).A limitation in the prior spectroscopic work is that the photoelectron peaks are assigned by comparing measured binding energies with computational results and that the molecular structures that underlie the calculations cannot not be independently determined in the experiments. This is compounded by the fact that theoretical calculation of charge-localized and charge-delocalized excited states is challenging. Widely used computational methods sometimes give unsatisfactory results (34), and even results from high-level computations can be controversial (35–37). This has led to interesting discussions about whether a stable charge-localized structure exists in the DMP cation at all and about the validity of different exchange functionals within density functional theory to study the charge localization in DMP molecules (34). Considering the general experimental and theoretical interest in this system, direct structural measurements would be invaluable. The current study uses ultrafast time-resolved X-ray scattering to observe the structural relaxation dynamics in DMP. This provides a wealth of information, making it possible to test key assumptions of the photoelectron study. Most importantly, we determine the molecular structures of the charge-localized and -delocalized excited states.     

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

Homologous flavoproteins from the photolyase (PHR)/cryptochrome (CRY) family use the FAD cofactor in PHRs to catalyze DNA repair and in CRYs to tune the circadian clock and control development. To help address how PHR/CRY members achieve these diverse functions, we determined the crystallographic structure of Arabidopsis thaliana (6-4) PHR (UVR3), which is strikingly (>65%) similar in sequence to human circadian clock CRYs. The structure reveals a substrate-binding cavity specific for the UV-induced DNA lesion, (6-4) photoproduct, and cofactor binding sites different from those of bacterial PHRs and consistent with distinct mechanisms for activities and regulation. Mutational analyses were combined with this prototypic structure for the (6-4) PHR/clock CRY cluster to identify structural and functional motifs: phosphate-binding and Pro-Lys-Leu protrusion motifs constricting access to the substrate-binding cavity above FAD, sulfur loop near the external end of the Trp electron-transfer pathway, and previously undefined C-terminal helix. Our results provide a detailed, unified framework for investigations of (6-4) PHRs and the mammalian CRYs. Conservation of key residues and motifs controlling FAD access and activities suggests that regulation of FAD redox properties and radical stability is essential not only for (6-4) photoproduct DNA repair, but also for circadian clock-regulating CRY functions. The structural and functional results reported here elucidate archetypal relationships within this flavoprotein family and suggest how PHRs and CRYs use local residue and cofactor tuning, rather than larger structural modifications, to achieve their diverse functions encompassing DNA repair, plant growth and development, and circadian clock regulation.     

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

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.     

Intrinsic functional architecture predicts electrically evoked responses in the human brain

Adaptive brain function is characterized by dynamic interactions within and between neuronal circuits, often occurring at the time scale of milliseconds. These complex interactions between adjacent and noncontiguous brain areas depend on a functional architecture that is maintained even in the absence of input. Functional MRI studies carried out during rest (R-fMRI) suggest that this architecture is represented in low-frequency (<0.1 Hz) spontaneous fluctuations in the blood oxygen level-dependent signal that are correlated within spatially distributed networks of brain areas. These networks, collectively referred to as the brain's intrinsic functional architecture, exhibit a remarkable correspondence with patterns of task-evoked coactivation as well as maps of anatomical connectivity. Despite this striking correspondence, there is no direct evidence that this intrinsic architecture forms the scaffold that gives rise to faster processes relevant to information processing and seizure spread. Here, we demonstrate that the spatial distribution and magnitude of temporally correlated low-frequency fluctuations observed with R-fMRI during rest predict the pattern and magnitude of corticocortical evoked potentials elicited within 500 ms after single-pulse electrical stimulation of the cerebral cortex with intracranial electrodes. Across individuals, this relationship was found to be independent of the specific regions and functional systems probed. Our findings bridge the immense divide between the temporal resolutions of these distinct measures of brain function and provide strong support for the idea that the low-frequency signal fluctuations observed with R-fMRI maintain and update the intrinsic architecture underlying the brain's repertoire of functional responses.     

Independent initiation of primary electron transfer in the two branches of the photosystem I reaction center

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with ∼100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P₇₀₀ (a pair of chlorophylls), and the tertiary acceptor, F_X (a Fe₄S₄ cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F_X, it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13¹-keto oxygen of either ec3_A (PsaA-Tyr696) or ec3_B (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2⁺ec3^- radical pair, followed by rapid reduction by P₇₀₀ in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.     

Quantification of hepatic functional capacity: a call for standardization

Reliable assessments of liver function are becoming increasingly important as more patients with surgically amenable liver disease are considered for treatment. Static markers of liver function are not sufficient to provide accurate assessments of hepatic function in order to risk stratify patients undergoing hepatic resection. Metabolic tests are dynamic indicators of liver function, but can be unreliable under certain conditions and thus difficult to make comparisons. Clearance tests avoid some of the pitfalls encountered during metabolic testing, but depend on hepatic blood flow and say little about hepatocyte function. Testing that combines imaging with measures of hepatocyte uptake may offer the most utility when planning surgical resections.     

Immunoreactive-somatostatin concentrations of the human stomach and mood state in patients with functional dyspepsia: A preliminary case–control study

Immunoreactive-somatostatin (ir-SS) concentrations of the gastric mucosa and mood state in patients with functional dyspepsia were examined. The subjects were 12 patients with upper abdominal discomfort, nausea and/or vomiting (motility disorder group) and 14 patients complaining of upper abdominal pain (ulcer-like disorder group) for more than a month without any organic upper-gastrointestinal tract disease proven by endoscopy. These patients were compared with either an age- and sex-matched group of asymptomatic outpatients without any organic disease (control group: n= 26) or to a group of patients with peptic ulcer (n= 19). Somatostatin concentrations of the stomach were measured by radio-immunoassay, and the mood state of each subject was assessed by Manifest Anxiety Scale (MAS) and Self-rating Depression Scale test. Immunoreactive-somatostatin concentrations of the gastric mucosa were significantly higher in the ulcer-like disorder group than in the peptic ulcer, motility disorder or control group, and gastric juice levels were higher in the ulcer-like disorder group. The psychometric tests showed that the motility disorder group was more depressive than the ulcer-like disorder group, but there were no differences between the motility disorder, ulcer-like disorder and peptic ulcer group in MAS scores or environmental factors. These results indicate that there may be two different subgroups in functional dyspepsia influenced by both ir-SS concentration of the stomach and/or mood state.     

Ultrafast large-amplitude relocation of electronic charge in ionic crystals

The interplay of vibrational motion and electronic charge relocation in an ionic hydrogen-bonded crystal is mapped by X-ray powder diffraction with a 100 fs time resolution. Photoexcitation of the prototype material KH₂PO₄ induces coherent low-frequency motions of the PO₄ tetrahedra in the electronically excited state of the crystal while the average atomic positions remain unchanged. Time-dependent maps of electron density derived from the diffraction data demonstrate an oscillatory relocation of electronic charge with a spatial amplitude two orders of magnitude larger than the underlying vibrational lattice motions. Coherent longitudinal optical and tranverse optical phonon motions that dephase on a time scale of several picoseconds, drive the charge relocation, similar to a soft (transverse optical) mode driven phase transition between the ferro- and paraelectric phase of KH₂PO₄.     

Prediction of functional disability by depressive state among community‐dwelling elderly in Japan: A prospective cohort study

Aim: We carried out a prospective cohort study to evaluate the risk factors of functional disability by depressive state. Methods: A total of 783 men and women, aged 70 years and over, participated in this study. We followed the participants in terms of the onset of functional disability by using a public long‐term care insurance database. The Geriatric Depression Scale (GDS) was used to measure depressive state. Age, sex, history of chronic disease, living alone, fall experience, cognitive impairment, instrumental activities of daily living (IADL), the Motor Fitness Scale (MFS), frequency of going out and social support at baseline were used as the main covariates. The Cox regression analysis was used to examine the difference in functional disability stratified according to depressive state. Results: The incidence of functional disability was 38 persons in the non‐depression group and 42 persons in the depression group (RR 2.34; 95% CI 1.46–3.79). The results of the depression group showed a significant difference in cognitive impairment (HR 3.51; 95% CI 1.39–8.85), MFS (HR 5.60; 95% CI 1.32–23.81) and IADL (HR 3.37; 95% CI 1.65–6.85). The results of the non‐depression group showed a significant difference in MFS (HR 2.97; 95% CI 1.47–6.96), and frequency of going out (HR 3.21; 95% CI 1.47–6.96). Conclusions: In conclusion, risk factors for functional disability were found to differ on the basis of whether or not community‐dwelling elderly individuals experience depressive state. The type of support offered must be based on whether or not depressive state is present. Geriatr Gerontol Int 2012; ??: ??–?? .     

Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle

Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP.P(i)) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP.P(i) state, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).     

Roles of multiple-proton transfer pathways and proton-coupled electron transfer in the reactivity of the bis-FeIV state of MauG

The high-valent state of the diheme enzyme MauG exhibits charge–resonance (CR) stabilization in which the major species is a bis-Fe^IV state with one heme present as Fe^IV=O and the other as Fe^IV with axial heme ligands provided by His and Tyr side chains. In the absence of its substrate, the high-valent state is relatively stable and returns to the diferric state over several minutes. It is shown that this process occurs in two phases. The first phase is redistribution of the resonance species that support the CR. The second phase is the loss of CR and reduction to the diferric state. Thermodynamic analysis revealed that the rates of the two phases exhibited different temperature dependencies and activation energies of 8.9 and 19.6 kcal/mol. The two phases exhibited kinetic solvent isotope effects of 2.5 and 2.3. Proton inventory plots of each reaction phase exhibited extreme curvature that could not be fit to models for one- or multiple-proton transfers in the transition state. Each did fit well to a model for two alternative pathways for proton transfer, each involving multiple protons. In each case the experimentally determined fractionation factors were consistent with one of the pathways involving tunneling. The percent of the reaction that involved the tunneling pathway differed for the two reaction phases. Using the crystal structure of MauG it was possible to propose proton–transfer pathways consistent with the experimental data using water molecules and amino acid side chains in the distal pocket of the high-spin heme.MauG (1) is a diheme enzyme that catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) (2) to complete the biosynthesis of its protein-derived cofactor (3) tryptophan tryptophylquinone (TTQ) (4). The hemes of MauG are unusual in several respects. One is a high-spin five-coordinate heme that is ligated by His35. The other is a low-spin six-coordinate heme with two ligands provided by His205 and Tyr294 (1, 5). The latter is, to our knowledge, the first example of natural His–Tyr ligation of a protein-bound heme cofactor, and the first example of Tyr ligation of a c-type heme. An intervening residue, Trp93, “connects” the two hemes (Fig. 1) via rapid electron transfer (ET) (6–9). A unique feature of MauG is that the oxidation of diferric MauG by H₂O₂, or of diferrous MauG by O₂, generates a high-valent bis-Fe^IV state (8) in which the high-spin heme is present as Fe^IV=O with the His35 ligand, and the other heme is present as Fe^IV with the His–Tyr axial ligation retained (5, 10, 11). Formation of the bis-Fe^IV state is accompanied by changes in the visible absorbance spectrum. One observes a decrease in intensity and shift of the Soret peak from 406 to 408 nm and appearance of minor peaks at 526 and 559 nm (Fig. 2) (9, 12).Open in a separate windowFig. 1.Diheme site of MauG. A portion of the crystal structure of the MauG-preMADH complex [Protein Data Bank (PDB) ID code 3L4M] is shown with MauG in pink, the MADH β-subunit in green, and the α subunit in blue. Shown in sticks are the hemes of MauG, the intervening Trp93, the three Met residues that are susceptible to autooxidation, the residues on preMADH that are modified by MauG, and Trp-199 which mediates ET from preMADH to bis-Fe^IV MauG. This figure was produced using PyMOL (www.pymol.org).Open in a separate windowFig. 2.Changes in the absorption spectrum of MauG caused by addition of H₂O₂ to diferric MauG. Spectra of MauG were recorded before (solid line) and after (dashed line) the addition of a stoichiometric amount of H₂O₂.The entire absorbance spectrum (A) is presented and the changes in the Soret region (B) and NIR region (C) are magnified.Despite being a highly potent oxidant, the bis-Fe^IV species displays extraordinary stability with a half-life of several minutes in the absence of its substrate (8). A basis for this stability was inferred from the observation of a near-infrared (NIR) electronic absorption feature centered at 950 nm that was observed in bis-Fe^IV MauG (Fig. 2C). This spectral feature is characteristic of a charge–resonance (CR) transition phenomenon (6, 9). A model was presented in which the CR occurs in the absence of direct heme–heme contact by ultrafast and reversible ET between the two high-valent hemes, via hopping through the intervening Trp93 residue (9). In this model the high-valent form of MauG comprises an ensemble of resonance structures including compound ES-like and compound I-like forms of the hemes, with the bis-Fe^IV as the dominant species.The catalytic mechanism of MauG is unusual in that the preMADH substrate does not make direct contact with either heme but instead binds to the surface of MauG several angstroms away (5). Catalysis requires long-range ET to bis-Fe^IV MauG from the residues on preMADH that are modified via a hole-hopping mechanism through Trp199 (13, 14), which resides at the MauG–preMADH interface (Fig. 1). Concomitant with this ET is the formation of free-radical intermediates on preMADH that go on to form the TTQ product (15). In the absence of preMADH, the autoreduction of the bis-Fe^IV redox state to the diferric state leads to inactivation of MauG (16). Analysis of the damaged MauG revealed that this process involves the oxidation of three Met residues (108, 114, and 116) which are located 7.5–15.2 Å from the high-spin heme iron (Fig. 1) (17).To further investigate the dynamic nature of the ensemble of resonance forms of MauG that comprise the high-valent state and the basis for its stability, temperature-dependence and kinetic solvent isotope effect (KSIE) studies were performed. These studies provide evidence for a redistribution within the ensemble of resonance structures before loss of CR stabilization of the high-valent redox state which is linked to the reduction to the diferric state. Thermodynamic analysis of the rates of reaction of these processes reveals that the rates of the initial redistribution of the ensemble of resonance structures and the subsequent loss of CR stabilization exhibit different dependencies on temperature. This accounts for the fact that the early phase is only observable at lower temperatures. Proton inventories of the KSIE indicate that the rates of both the initial redistribution of the ensemble of high-valent species and the loss of CR stabilization are rate-limited by multiple proton-transfer (PT) steps involving two alternative pathways. The likely pathways are identified from the crystal structure of MauG.     

Precuneus shares intrinsic functional architecture in humans and monkeys

Evidence from macaque monkey tracing studies suggests connectivity-based subdivisions within the precuneus, offering predictions for similar subdivisions in the human. Here we present functional connectivity analyses of this region using resting-state functional MRI data collected from both humans and macaque monkeys. Three distinct patterns of functional connectivity were demonstrated within the precuneus of both species, with each subdivision suggesting a discrete functional role: (i) the anterior precuneus, functionally connected with the superior parietal cortex, paracentral lobule, and motor cortex, suggesting a sensorimotor region; (ii) the central precuneus, functionally connected to the dorsolateral prefrontal, dorsomedial prefrontal, and multimodal lateral inferior parietal cortex, suggesting a cognitive/associative region; and (iii) the posterior precuneus, displaying functional connectivity with adjacent visual cortical regions. These functional connectivity patterns were differentiated from the more ventral networks associated with the posterior cingulate, which connected with limbic structures such as the medial temporal cortex, dorsal and ventromedial prefrontal regions, posterior lateral inferior parietal regions, and the lateral temporal cortex. Our findings are consistent with predictions from anatomical tracer studies in the monkey, and provide support that resting-state functional connectivity (RSFC) may in part reflect underlying anatomy. These subdivisions within the precuneus suggest that neuroimaging studies will benefit from treating this region as anatomically (and thus functionally) heterogeneous. Furthermore, the consistency between functional connectivity networks in monkeys and humans provides support for RSFC as a viable tool for addressing cross-species comparisons of functional neuroanatomy.     

Concerted electron-proton transfer in the optical excitation of hydrogen-bonded dyes

The simultaneous, concerted transfer of electrons and protons--electron-proton transfer (EPT)--is an important mechanism utilized in chemistry and biology to avoid high energy intermediates. There are many examples of thermally activated EPT in ground-state reactions and in excited states following photoexcitation and thermal relaxation. Here we report application of ultrafast excitation with absorption and Raman monitoring to detect a photochemically driven EPT process (photo-EPT). In this process, both electrons and protons are transferred during the absorption of a photon. Photo-EPT is induced by intramolecular charge-transfer (ICT) excitation of hydrogen-bonded-base adducts with either a coumarin dye or 4-nitro-4'-biphenylphenol. Femtosecond transient absorption spectral measurements following ICT excitation reveal the appearance of two spectroscopically distinct states having different dynamical signatures. One of these states corresponds to a conventional ICT excited state in which the transferring H(+) is initially associated with the proton donor. Proton transfer to the base (B) then occurs on the picosecond time scale. The other state is an ICT-EPT photoproduct. Upon excitation it forms initially in the nuclear configuration of the ground state by application of the Franck-Condon principle. However, due to the change in electronic configuration induced by the transition, excitation is accompanied by proton transfer with the protonated base formed with a highly elongated (+)H ─ B bond. Coherent Raman spectroscopy confirms the presence of a vibrational mode corresponding to the protonated base in the optically prepared state.     

Resting state functional connectivity as a predictor of brief intervention response in adults with alcohol use disorder: A preliminary study

Background

Brief interventions for alcohol use disorder (AUD) are generally efficacious, albeit with variability in response. Resting state functional connectivity (rsFC) may characterize neurobiological indicators that predict the response to brief interventions and is the focus of the current investigation.

Materials and Methods

Forty-six individuals with AUD (65.2% female) completed a resting state functional magnetic resonance imaging (fMRI) scan immediately followed by a brief intervention aimed at reducing alcohol consumption. Positive clinical response was defined as a reduction in alcohol consumption by at least one World Health Organization (WHO) risk drinking level at 3-month follow-up. rsFC was analyzed using seed-to-voxel analysis with seed regions from four networks: salience network, reward network, frontoparietal network, and default mode network.

Results

At baseline, responders had greater rsFC between the following seed regions in relation to voxel-based clusters than non-responders: (i) anterior cingulate cortex (ACC) in relation to left postcentral gyrus and right supramarginal gyrus (salience network); (ii) right posterior parietal cortex in relation to right ventral ACC (salience network); (iii) right interior frontal gyrus (IFG) pars opercularis in relation to right cerebellum and right occipital fusiform gyrus (frontoparietal); and (iv) right primary motor cortex in relation to left thalamus (default mode). Lower rsFC in responders vs. nonresponders was seen between the (i) right rostral prefrontal cortex in relation to left IFG pars triangularis (frontoparietal); (ii) right IFG pars triangularis in relation to right cerebellum (frontoparietal); (iii) right IFG pars triangularis in relation to right frontal eye fields and right angular gyrus (frontoparietal); and (iv) right nucleus accumbens in relation to right orbital frontal cortex and right insula (reward).

Conclusions

Resting state functional connectivity in the frontoparietal, salience, and reward networks predicts the response to a brief intervention in individuals with AUD and could reflect greater receptivity or motivation for behavior change.

     

Rational modulation of conformational fluctuations in adenylate kinase reveals a local unfolding mechanism for allostery and functional adaptation in proteins

Elucidating the complex interplay between protein structure and dynamics is a prerequisite to an understanding of both function and adaptation in proteins. Unfortunately, it has been difficult to experimentally decouple these effects because it is challenging to rationally design mutations that will either affect the structure but not the dynamics, or that will affect the dynamics but not the structure. Here we adopt a mutation approach that is based on a thermal adaptation strategy observed in nature, and we use it to study the binding interaction of Escherichia coli adenylate kinase (AK). We rationally design several single-site, surface-exposed glycine mutations to selectively perturb the excited state conformational repertoire, leaving the ground-state X-ray crystallographic structure unaffected. The results not only demonstrate that the conformational ensemble of AK is significantly populated by a locally unfolded state that is depopulated upon binding, but also that the excited-state conformational ensemble can be manipulated through mutation, independent of perturbations of the ground-state structures. The implications of these results are twofold. First, they indicate that it is possible to rationally design dynamic allosteric mutations, which do not propagate through a pathway of structural distortions connecting the mutated and the functional sites. Secondly and equally as important, the results reveal a general strategy for thermal adaptation that allows enzymes to modulate binding affinity by controlling the amount of local unfolding in the native-state ensemble. These findings open new avenues for rational protein design and fundamentally illuminate the role of local unfolding in function and adaptation.