Spatiotemporal reaction kinetics of an ultrafast photoreaction pathway visualized by time-resolved liquid x-ray diffraction

We have studied the reaction dynamics for HgI(2) in methanol by using time-resolved x-ray diffraction (TRXD). Although numerous time-resolved spectroscopic studies have provided ample information about the early dynamics of HgI(2), a comprehensive reaction mechanism in the solution phase spanning from picoseconds up to microseconds has been lacking. Here we show that TRXD can provide this information directly and quantitatively. Picosecond optical pulses triggered the dissociation of HgI(2), and 100-ps-long x-ray pulses from a synchrotron probed the evolving structures over a wide temporal range. To theoretically explain the diffracted intensities, the structural signal from the solute, the local structure around the solute, and the hydrodynamics of bulk solvents were considered in the analysis. The results in this work demonstrate that the determination of transient states in solution is strongly correlated with solvent energetics, and TRXD can be used as an ultrafast calorimeter. It also is shown that a manifold of structural channels can be resolved at the same time if the measurements are accurate enough and that global analysis is applied. The rate coefficients for the reactions were obtained by fitting our model against the experimental data in one global fit including all q-values and time delays. The comparison between all putative reaction channels confirms that two-body dissociation is the dominant dissociation pathway. After this primary bond breakage, two parallel channels proceed. Transient HgI associates nongeminately with an iodine atom to form HgI(2), and I(2) is formed by nongeminate association of two iodine atoms.     

Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction.     

Observations of reaction intermediates and the mechanism of aldose-ketose interconversion by D-xylose isomerase.

Crystallographic studies of D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) incubated to equilibrium with substrate/product mixtures of xylose and xylulose show electron density for a bound intermediate. The accumulation of this bound intermediate shows that the mechanism is a non-Michaelis type. Carrell et al. [Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch, D. & Biellmann, J.-F. (1989) Proc. Natl. Acad. Sci. USA 86, 4440-4444] and the present authors studied crystals of the enzyme-substrate complex under different conditions and made different interpretations of the substrate density, leading to different conclusions about the enzyme mechanism. All authors agree that the bound intermediate of the sugar is in an open-chain form. It is suggested that the higher-temperature study of Carrell et al. may have produced an equilibrium of multiple states, whose density fits poorly to the open-chain substrate, and led to incorrect interpretation. The two groups also bound different closed-ring sugar analogues to the enzyme, but these analogues bind differently. A possible explanation consistent with all the data is that the enzyme operates by a hydride shift mechanism.     

Protein folding kinetics and thermodynamics from atomistic simulation

Advances in simulation techniques and computing hardware have created a substantial overlap between the timescales accessible to atomic-level simulations and those on which the fastest-folding proteins fold. Here we demonstrate, using simulations of four variants of the human villin headpiece, how simulations of spontaneous folding and unfolding can provide direct access to thermodynamic and kinetic quantities such as folding rates, free energies, folding enthalpies, heat capacities, Φ-values, and temperature-jump relaxation profiles. The quantitative comparison of simulation results with various forms of experimental data probing different aspects of the folding process can facilitate robust assessment of the accuracy of the calculations while providing a detailed structural interpretation for the experimental observations. In the example studied here, the analysis of folding rates, Φ-values, and folding pathways provides support for the notion that a norleucine double mutant of villin folds five times faster than the wild-type sequence, but following a slightly different pathway. This work showcases how computer simulation has now developed into a mature tool for the quantitative computational study of protein folding and dynamics that can provide a valuable complement to experimental techniques.     

Structural kinetics of myosin by transient time-resolved FRET

For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)(2)FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)(2)FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)(2)FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.     

Mechanistic studies of glutamine synthetase from Escherichia coli: kinetic evidence for two reaction intermediates in biosynthetic reaction.

Fast reaction techniques were used to study the kinetics of protein fluorescence intensity changes that are associated with the reactions of unadenylylated Escherichia coli glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] with its substrates. It was established that the synthesis of glutamine occurs by a stepwise mechanism. During the catalytic process two fluorometrically distinct intermediates were observed. Both forward and reverse rate constants which lead to the formation and consumption of these intermediates were evaluated. The catalytic rate constant, kc, which was calculated from these rate constants agrees well with the values of kc which were determined by direct measurement of the overall biosynthetic activities by means of stopped-flow technique or the steady-state assay method.     

Protein folding: How the mechanism of GroEL action is defined by kinetics

We propose a mechanism for the role of the bacterial chaperonin GroEL in folding proteins. The principal assumptions of the mechanism are (i) that many unfolded proteins bind to GroEL because GroEL preferentially binds small unstructured regions of the substrate protein, (ii) that substrate protein within the cavity of GroEL folds by the same kinetic mechanism and rate processes as in bulk solution, (iii) that stable or transient complexes with GroEL during the folding process are defined by a kinetic partitioning between formation and dissociation of the complex and the rate of folding and unfolding of the protein, and (iv) that dissociation from the complex in early stages of folding may lead to aggregation but dissociation at a late stage leads to correct folding. The experimental conditions for refolding may play a role in defining the function of GroEL in the folding pathway. We propose that the role of GroES and MgATP, either binding or hydrolysis, is to regulate the association and dissociation processes rather than affecting the rate of folding.     

Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures

Here we present a meta-analysis of a large collection of static structures of a protein in the Protein Data Bank in order to extract the progression of structural events during protein function. We apply this strategy to the homodimeric hemoglobin HbI from Scapharca inaequivalvis. We derive a simple dynamic model describing how binding of the first ligand in one of the two chemically identical subunits facilitates a second binding event in the other partner subunit. The results of our ultrafast time-resolved crystallographic studies support this model. We demonstrate that HbI functions like a homodimeric mechanical device, such as pliers or scissors. Ligand-induced motion originating in one subunit is transmitted to the other via conserved pivot points, where the E and F' helices from two partner subunits are "bolted" together to form a stable dimer interface permitting slight relative rotation but preventing sliding.     

Ensemble molecular dynamics yields submillisecond kinetics and intermediates of membrane fusion

Lipid membrane fusion is critical to cellular transport and signaling processes such as constitutive secretion, neurotransmitter release, and infection by enveloped viruses. Here, we introduce a powerful computational methodology for simulating membrane fusion from a starting configuration designed to approximate activated prefusion assemblies from neuronal and viral fusion, producing results on a time scale and degree of mechanistic detail not previously possible to our knowledge. We use an approach to the long time scale simulation of fusion by constructing a Markovian state model with large-scale distributed computing, yielding an understanding of fusion mechanisms on time scales previously impossible to simulate to our knowledge. Our simulation data suggest a branched pathway for fusion, in which a common stalk-like intermediate can either rapidly form a fusion pore or remain in a metastable hemifused state that slowly forms fully fused vesicles. This branched reaction pathway provides a mechanistic explanation both for the biphasic fusion kinetics and the stable hemifused intermediates previously observed experimentally. Our distributed computing and Markovian state model approaches provide sufficient sampling to detect rare transitions, a systematic process for analyzing reaction pathways, and the ability to develop quantitative approximations of reaction kinetics for fusion.     

Fluctuation spectroscopy: determination of chemical reaction kinetics from the frequency spectrum of fluctuations

The kinetic parameters of a chemical reaction were obtained from analysis of the frequency spectrum of the fluctuations (i.e., “noise”) in the concentrations of the reactants. In “fluctuation spectroscopy,” no external perturbation is applied and the system remains in macroscopic chemical equilibrium during the experiment. Results obtained by this method for the dissociation reaction of beryllium sulfate agree well with those obtained by relaxation methods in which the approach to equilibrium is analyzed. Other noise sources not originating from a chemical reaction were observed and analyzed. The most prominent of these arose from the flow of an electrolyte through a capillary. The method of fluctuation spectroscopy should be applicable to problems of physical, chemical, and biological interest.     

Chemistry of low-valent molybdenum phosphite complexes: Models of seven-coordinate reaction intermediates

A simple synthesis of the zerovalent complex Mo[P(OCH₃)₃]₆ has been devised from a potassium reduction of MoCl₄(tetrahydrofuran)₂ followed by reaction with trimethyl phosphite at 70°. Protonation of this octahedral complex gave only low yields of the expected seven-coordinate hydride, HMo[P(OCH₃)₃]₆⁺. The major product was an octahedral nonhydridic cation, Mo[P(OCH₃)₃]₅P(OCH₃)₂⁺, derived from proton cleavage of the P—O phosphite ester bond. This octahedral cation was stereochemically nonrigid, apparently through facile methoxy group migration. Close packing by methoxy groups in this fluxional cation was established through an x-ray crystallographic study of Mo[P(OCH₃)₃]₅P(OCH₃)₂⁺-PF₆^-. Extended reaction of trifluoroactic acid with Mo[P(OCH₃)₃]₆ yielded the seven-coordinate hydride, HMo[P(OCH₃)₃]₄(O₂CCF₃), which was near pentagonal bipyramidal and was stereochemically nonrigid.     

Protein hydration in solution: Experimental observation by x-ray and neutron scattering

The structure of the protein–solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H₂O and D₂O solutions. The analysis of the protein–solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density ≈10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces.     

Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism

Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 A; R = 22.2%; R(free) = 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 A; R = 24.1%; R(free) = 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.     

Millisecond time-resolved changes in x-ray reflections from contracting muscle during rapid mechanical transients, recorded using synchrotron radiation.

Low-angle x-ray diffraction diagrams have been recorded from frog sartorius muscles by using synchrotron radiation as a high-intensity x-ray source. This has enabled changes in some of the principal reflections of interest to be followed with a time resolution of 1 ms, during small but very rapid length changes imposed on a contracting muscle. The 143-A meridional reflection, which is believed to arise from a repeating pattern of myosin cross-bridges along the length of the muscle, shows large changes in intensity in these circumstances. During both rapid releases and rapid stretches, by amounts that produce a translation of actin and myosin filaments past each other by about 100 A and that are completed in about a millisecond (i.e., before significant cross-bridge detachment would be expected), an almost synchronous decrease in 143-A intensity occurs, by 50% or more. This is followed, in the case of quick releases, by a rapid partial recovery of intensity lasting 5--6 ms (which may represent cross-bridge release and reattachment) and then by a more gradual return to the normal isometric value. Quick stretches show only the slower return of intensity. Immediately after the length change, the initial drop in 143-A intensity can be reversed if the release (or stretch) is reversed. These changes provide evidence of a more direct kind than has hitherto been available that the active sliding of actin filaments past myosin filaments during contraction is produced by longitudinal movement of attached cross-bridges.     

Catalytic mechanism of quinoprotein methanol dehydrogenase: A theoretical and x-ray crystallographic investigation

The catalytic mechanism of the reductive half reaction of the quinoprotein methanol dehydrogenase (MDH) is believed to proceed either through a hemiketal intermediate or by direct transfer of a hydride ion from the substrate methyl group to the cofactor, pyrroloquinoline quinone (PQQ). A crystal structure of the enzyme-substrate complex of a similar quinoprotein, glucose dehydrogenase, has recently been reported that strongly favors the hydride transfer mechanism in that enzyme. A theoretical analysis and an improved refinement of the 1.9-A resolution crystal structure of MDH from Methylophilus methylotrophus W3A1 in the presence of methanol, reported earlier, indicates that the observed tetrahedral configuration of the C-5 atom of PQQ in that study represents the C-5-reduced form of the cofactor and lends support for a hydride transfer mechanism for MDH.     

Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe³⁺ to activate O₂ and catecholic substrates for reaction. The inability of Fe³⁺ to directly bind O₂ presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe³⁺ species, and the anhydride-Fe³⁺ intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe²⁺-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe²⁺ intermediate that could bind O₂ are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.Most ring-cleaving dioxygenases belong to the nonheme mononuclear iron-containing enzyme family, many members of which play critical roles in the aerobic biodegradation of natural and synthetic aromatic compounds (1–5). Ring cleavage by these enzymes involves fission of the O–O bond of dioxygen and incorporation of both atoms into the product (6). Two major classes of catechol ring-cleaving dioxygenases have been described. These differ in the mode of cleavage and the oxidation state of the iron cofactor: the Fe²⁺-using extradiol dioxygenases (EDOs) cleave adjacent to the vicinal hydroxyl functions of catechols (meta cleavage) while intradiol dioxygenases (IDOs) use a Fe³⁺ cofactor and cleave between the hydroxyl functions (ortho cleavage). The Fe³⁺ in the active site of IDOs will not bind O₂ and no direct spectroscopic evidence for redox cycling has been obtained (1). This means that IDOs must activate O₂ using a unique mechanism, which differs from that of the much more common Fe²⁺ containing oxygenases that readily form an Fe–O₂ complex after the substrate binds in the active site (7–9).The archetypal IDO is protocatechuate 3,4-dioxygenase (3,4-PCD), which catalyzes cleavage of its native substrate (3,4-dihydroxybenzoate, PCA) to yield β-carboxy-cis,cis-muconate, thereby funneling aromatic substrates into the β-ketoadipate pathway (10). Much of our mechanistic knowledge of IDOs comes from kinetic, spectroscopic, and crystallographic studies of 3,4-PCD, catechol 1,2-dioxygenase (11–17), and bio-inspired model compounds (18, 19). The 3,4-PCD coordinates the Fe³⁺ in a 2-His, 2-Tyr ligand set that is conserved in IDOs (11). The tyrosine ligands contribute several ligand-to-metal-charge transfer bands (LMCTs) in the visible, which give IDOs a burgundy color that can be used to monitor the reaction (16). In 3,4-PCD, the Fe³⁺ is coordinated by axial ligands Y447 and H462 and equatorial ligands H460, Y408, and solvent-derived hydroxide (Fig. 1) (11).Open in a separate windowFig. 1.Proposed intermediates in the reaction of 3,4-PCD with substrate and O₂. R represents either COOH (PCA) or F (4FC). 4FC is bound in the equatorial plane rather than the axial plane as shown in 2. It may rotate to the axial plane to react. The π_op-sym highest occupied molecular orbital of substrate is proposed to be the source of the reducing equivalents (15).The proposed intermediates for the reaction of 3,4-PCD with PCA and O₂ are shown in Fig. 1 and designated 1–8 (1, 13, 15, 17, 20–23). First, PCA binds to the metal of the resting enzyme 1 in a multistep process leading to a complex 2 where both hydroxyls are ionized and the ring of PCA is axially oriented. Formation of 2 results in dissociation of Y447 from the metal, forming a 5-coordinate (5C) complex with a vacant site trans to H460. O₂ reacts with 2 in a concerted process 3, generating a Fe³⁺-alkylperoxo species 4. Spectroscopic and computational studies suggest that catalysis requires a physical shift of a transient six-coordinate (6C) alkyperoxo 4 to a 5C species 5, opening the axial site trans to H462. Protonation of the peroxo moiety 6 drives O–O bond scission and migration of one oxygen atom into the substrate ring to form an anhydride 7. Attack of the iron-bound hydroxide on the anhydride cleaves the substrate ring to form product complex 8, which, upon release, regenerates state 1. Transient kinetic studies have detected intermediates throughout the catalytic cycle, but the kinetics of their interconversion have not allowed any intermediate between the substrate and product complexes to be trapped for detailed characterization (12, 13, 24, 25).The use of alternative substrates has been a successful means to detect and trap intermediates in enzyme reaction cycles in which the native reaction proceeds too quickly or with unfavorable interconversion-rate constants (26). A powerful extension of this approach involves performing catalysis in enzyme crystals to trap reactive species for characterization using X-ray crystallography, which has been successfully applied to a few systems (27–31). This approach was used by our laboratory to identify and characterize four distinct oxygenated intermediates in the catalytic cycle of the EDO homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) (29, 30). Here, we have extended this in crystallo approach to the IDO 3,4-PCD as it turns over the alternative substrate 4-fluorocatechol (4FC), allowing determination of the structures of two intermediates following O₂ addition. These intermediates provide a direct view of the key steps in the oxygen activation and reaction cycle of IDOs. Insight is also gained into one of the longstanding questions in oxygenase research: the basis for cleavage site specificity in the ring-cleaving dioxygenase family.     

On the mechanism of genetic recombination: the maturation of recombination intermediates.

DNA molecules of the plasmid ColEl are normally recovered from wild-type cells as a set of monomer- and multimer-size rings. The data of this paper show that the multimer-size species are a product of genetic recombination. Multimer rings do not arise after transfection of purified monomers into bacterial host cells lacking a functional recA recombination system. Analogously, purified dimers, trimers, and tetramers, transfected into recA- cells, can replicate, but are constrained to remain in those conformations. Only upon transfection into rec+ cells can they regenerate the full spectrum of monomer- and multimer-size species. In this paper we trace the flow of genetic information from the monomer to the multimer state and back again under the guidance of the recA recombination system. The formation of multimer-size DNA rings is discussed as a natural consequence of the maturation of a Holliday recombination intermediate formed between two monomer plasmid genomes.