Theoretical study of the unusual protonation properties of the active site cysteines in thioredoxin

The thiol/disulfide oxidoreductases of the thioredoxin family have, in the active site, two cysteines that can be in a reduced or an oxidized form. One of the cysteines in the reduced state is deprotonated, and it is called nucleophilic cysteine. The pK(a) of this cysteine is different from that of a normal cysteine and varies widely among the different enzymes of this family. However, the factors responsible for the different degrees of stabilization of nucleophilic cysteine thiolate are not fully understood. Here, we have studied the well-known hypothesis of proton sharing between the active site thiols by performing a linear transit scan for the transfer of the proton between the active site cysteines. We used a two-layered (DFT/MM) ONIOM formalism, with the active site region treated at the B3LYP/6-31+G(d) level and the remains of the protein treated with the Amber Parm94 force field. The solvation free energy was accounted for with a continuum solvent model, by solving the Poisson-Boltzmann equation using the program Delphi. We have obtained excellent agreement with the experimental data available in the literature. Besides refuting the proton sharing hypothesis, our results include a value of 14.0 for the pK(a) of the buried cysteine, a quantity that has not been possible to obtain experimentally but which has been proven to be higher than 11. Additionally, this study also provides detailed information on the very interesting and so far unknown fact that the contribution of the enzymatic structure (8.3 kcal/mol) prevails in relation to that of the solvent (0.60 kcal/mol) concerning the differential stabilization of the active site thiolates.     

Spectroscopic evidence for site specific chemistry at a unique iron site of the [4Fe-4S] cluster in ferredoxin:thioredoxin reductase

Ferredoxin:thioredoxin reductase (FTR) catalyzes the reduction of the disulfide in thioredoxin in two one-electron steps using an active site comprising a [4Fe-4S] in close proximity to a redox active disulfide. M?ssbauer spectroscopy has been used to investigate the ligation and electronic properties of the [4Fe-4S] cluster in as-prepared FTR which has the active-site disulfide intact and in the N-ethylmaleimide (NEM)-modified form which provides a stable analogue of the one-electron-reduced heterodisulfide intermediate and has one of the cysteines of the active-site disulfide alkylated with NEM. The results reveal novel site-specific cluster chemistry involving weak interaction of the active-site disulfide with a unique Fe site of the [4Fe-4S]2+ cluster in the resting enzyme and cleavage of the active-site disulfide with concomitant coordination of one of the cysteines to yield a [4Fe-4S]3+ cluster with a five-coordinate Fe site ligated by two cysteine residues in the NEM-modified enzyme. The results provide molecular-level insight into the catalytic mechanism of FTR and other Fe-S-cluster-containing disulfide reductases, and suggest a possible mechanism for the reductive cleavage of S-adenosylmethionine by the radical SAM family of Fe-S enzymes.     

Role of the variable active site residues in the function of thioredoxin family oxidoreductases

The enzymes of the thioredoxin family fulfill a wide range of physiological functions. Although they possess a similar CXYC active site motif, with identical environment and stereochemical properties, the redox potential and pK(a) of the cysteine pair varies widely across the family. As a consequence, each family member promotes oxidation or reduction reactions, or even isomerization reactions. The analysis of the three-dimensional structures gives no clues to identify the molecular source for the different active site properties. Therefore, we carried out a set of quantum mechanical calculations in active site models to gain more understanding on the elusive molecular-level origin of the differentiation of the properties across the family. The obtained results, together with earlier quantum mechanical calculations performed in our laboratories, gave rise to a consistent line of evidence, which points to the fact that both active site cysteines play an important role in the differentiation. In contrary to what was assumed, differentiation is not achieved through a different stabilization of the solvent exposed cysteine but, instead, through a fine tuning of the nucleophilicity of both active site cysteines. Reductant enzymes have both cysteine thiolates poorly stabilized, oxidant proteins have both cysteine thiolates highly stabilized, and isomerases have one thiolate (solvent exposed) poorly stabilized and the other (buried) thiolate highly stabilized. The feasibility of shifting the chemical equilibrium toward oxidation, reduction, or isomerization only through subtle electrostatic effects is quite unusual, and it relies on the inherent thermoneutrality of the catalytic steps carried out by a set of chemically equivalent entities all of which are cysteine thiolates. Such pattern of stabilization/destabilization, detected in our calculations is fully consistent with the observed physiological roles of this family of enzymes.     

Ab initio and QM/MM study of electron addition on the disulfide bond in thioredoxin

Thioredoxin controls the intracellular redox potential through a disulfide/dithiol couple. Under conditions of oxidative stress, this protein functions via one-electron exchange, in which formation of the disulfide radical anion occurs. Combined quantum mechanical (QM) and molecular mechanical (MM) calculations using two- and three-level ONIOM schemes were performed on the thioredoxin (Trx) protein of Chlamydomonas reinhardtii in its oxidized-disulfide and one-electron-reduced forms. In both cases, the active site disulfide moiety was described at the MP2(fc)/6-31+G(d) level, and larger regions of varying sizes around the active site were described at the B3LYP/6-31+G(d) level. The remainder of the 112 residues and 33 water molecules of the crystal structure (PDB entry 1EP7) were described by the AMBER force field. Adiabatic electron affinities were calculated for the disulfide bond in all systems. Separate QM or QM/QM calculations were performed on the QM regions to establish the role of the remainder of the protein on the active site properties. The radical anion species becomes more stable as the number of amide groups in the vicinity increases. One-electron reduction potentials were calculated for the small molecule models, and approximated for the protein for which the values are similar to the experimental one (approximately 0 V). This high reduction potential is due to interaction with the charged end of Lys40, as indicated by mutation in silico to norleucine. The inclusion of the protonated Asp30 side chain and a water molecule in the QM region leads to an increase in the electron affinity. Proton transfer from the Asp30 side chain to the Cys39 sulfur in the radical anion species is strongly disfavored. The radical anion is more stable than the protonated form, which is consistent with experimental results.     

Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism

Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and M?ssbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.     

A small-molecule catalyst of protein folding in vitro and in vivo

BACKGROUND: The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (Eo'). RESULTS: A synthetic small-molecule dithiol, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an Eo' value of -0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo. CONCLUSIONS: A small-molecule dithiol with a low thiol pKa value and high disulfide Eo' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo.     

Structural and functional diversity of glutaredoxins in yeast

Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a complex family of proteins with a diversity of enzymatic and functional properties. Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate mixed disulfides between glutathione and cysteine protein residues. They could act regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such a role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membrane-associated glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins, which may act as redox regulators at the early stages of the protein secretory machinery.     

Biomimetic studies on selenoenzymes: modeling the role of proximal histidines in thioredoxin reductases

The roles of built-in thiol cofactors and the basic histidine (His) residues in the active site of mammalian thioredoxin reductases (TrxRs) are described with the help of experimental and density functional theory calculations on small-molecule model compounds. The reduction of selenenyl sulfides by thiols in selenoenzymes such as glutathione peroxidase (GPx) and TrxR is crucial for the regeneration of the active site. Experimental as well as theoretical studies were carried out with model selenenyl sulfides to probe their reactivity toward incoming thiols. We have shown that the nucleophilic attack of thiols takes place at the selenium center in the selenenyl sulfides. These thiol exchange reactions would hamper the regeneration of the active species selenol. Therefore, the basic His residues are expected to play crucial roles in the selenenyl sulfide state of TrxR. Our model study with internal amino groups in the selenenyl sulfide state reveals that the basic His residues may play important roles by deprotonating the thiol moiety in the selenenic acid state and by interacting with the sulfur atom in the selenenyl sulfide state to facilitate the nucleophilic attack of thiol at sulfur rather than at selenium, thereby generating the catalytically active species selenol. This model study also suggests that the enzyme may use the internal cysteines as cofactors to overcome the thiol exchange reactions.     

On the origin of the stabilization of the zwitterionic resting state of cysteine proteases: a theoretical study

Papain-like cysteine proteases are ubiquitous proteolytic enzymes. The protonated His199/deprotonated Cys29 ion pair (cathepsin B numbering) in the active site is essential for their proper functioning. The presence of this ion pair stands in contrast to the corresponding intrinsic residue p K a values, indicating a strong influence of the enzyme environment. In the present work we show by molecular dynamics simulations on quantum mechanical/molecular mechanical (QM/MM) potentials that the ion pair is stabilized by a complex hydrogen bond network which comprises several amino acids situated in the active site of the enzyme and 2-4 water molecules. QM/MM reaction path computations for the proton transfer from His199 to the thiolate of the Cys29 moiety indicate that the ion pair is about 32-36 kJ mol (-1) more stable than the neutral form if the whole hydrogen bonding network is active. Without any hydrogen bonding network the ion pair is predicted to be significantly less stable than the neutral form. QM/MM charge deletion analysis and QM model calculations are used to quantify the stabilizing effect of the active-site residues and the L1 helix in favor of the zwitterionic form. The active-site water molecules contribute about 30 kJ mol (-1) to the overall stabilization. Disruption of the hydrogen bonding network upon substrate binding is expected to enhance the nucleophilic reactivity of the thiolate.     

Identification of redox sensitive thiols of protein disulfide isomerase using isotope coded affinity technology and mass spectrometry

Regulation of the redox state of protein disulfide isomerase (PDI) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDI. Human PDI contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDI while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDI under various conditions.     

Mechanism of thioredoxin-catalyzed disulfide reduction. Activation of the buried thiol and role of the variable active-site residues

Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in other proteins. We have theoretically explored this reaction mechanism, both in the gas phase and in water, using density functional theory. The mechanism of disulfide reduction involves two consecutive thiol-disulfide exchange reactions, that is, nucleophilic substitutions at sulfur (S(N)2@S): first, by one Trx cysteine-thiolate group (Cys-32) at a sulfur atom of the disulfide substrate and, second, by the other Trx cysteine-thiolate group (the buried thiol of Cys-35) at the sulfur atom of the first Trx cysteine. We have investigated the intrinsic nature of such S(N)2@S substitution using the simple CH3S(-) + CH3SSCH3 model and how it is affected by solvation in aqueous solution. Next, we have examined how the behavior of the elementary S(N)2@S steps changes in the more realistic enzyme-substrate model CGPC + CH3SSCH3, which contains the active-site of Trx. In all model reactions, solvation turns the hypervalent trisulfide anion (i.e., the S(N)2@S transition species) from a stable complex into a transition state. Importantly, our analyses suggest that the deprotonation of the buried thiol (which is required before the latter can enter into the second S(N)2@S step) is done by the leaving group evolving from the first S(N)2@S step. Finally, molecular dynamics (MD) simulations, in the gas phase and in water, of CGPC, CGGC, and the corresponding wild-type Trx and P34G Trx show that the activity of the thioredoxin active-site motif (CXYC) is determined not only by the structural rigidity associated with the particular variable residues (XY) but also by the number of amide N-H groups. The latter are involved in the stabilization of the Cys-32 thiolate and thus affect the acidity and nucleophilicity of this residue.     

Defining the plant disulfide proteome

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.     

Zinc-bound thiolate-disulfide exchange: a strategy for inhibiting metallo-beta-lactamases

The mononuclear zinc thiolate complexes [(Tp(PhMe))Zn(S-R)], where Tp(PhMe) is hydrotris((3-methyl-5-phenyl)pyrazolyl)borate and (S-R) is benzyl thiolate, 4-nitrophenylthiolate, 4-trifluoromethylphenylthiolate, 4-chlorophenylthiolate, phenylthiolate, 2-methylphenylthiolate, 4-methylphenylthiolate, 4-methoxyphenylthiolate, or 4-hydroxyphenylthiolate, were synthesized. Representative members of the class were also characterized structurally. The benzyl thiolate complex undergoes a thiolate-disulfide exchange reaction with a variety of diphenyl and dipyridyl disulfides. Kinetic studies revealed that the reaction shows saturation behavior in both complex and disulfide for most of the disulfides studied. Combined with studies of the lability of the coordinated thiolate, a mechanism is proposed where the reactive species is the zinc-coordinated thiolate. When the free benzyl thiol was allowed to react with the same disulfides, the reaction was slower by a factor of 20-200 than that for the zinc-thiolate complex, depending on the particular disulfide employed. Since most metallo-beta-lactamases contain one or more cysteine residues, the one in the active site being coordinated to zinc, the present study was extended to examine whether disulfides can be used as inhibitors of these enzymes by selective oxidation of the metal-bound cysteine. Several disulfides allowed to react with metallo-beta-lactamase CcrA from Bacteroides fragilis were moderate to potent irreversible inhibitors of the enzyme.     

Application of Fragment‐Based Screening to the Design of Inhibitors of Escherichia coli DsbA

The thiol‐disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram‐negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell‐based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.     

Solid-state NMR study of the charge-transfer complex between ubiquinone-8 and disulfide bond generating membrane protein DsbB

Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB, and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ~500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge-transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (~20 ppm), methoxys (~60 ppm), olefin carbons (120-140 ppm), and carbonyls (150-160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge-transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.     

Photophysical properties of ruthenium bipyridine (4-carboxylic acid-4'-methyl-2,2'-bipyridine) complexes and their acid-base chemistry

Photophysical properties such as absorption and emission spectra, lifetimes, and redox potentials of eight ruthenium complexes, Ru(LL)2(MebpyCOOH)2+, where LL represents bpy, phen, Me2bpy, Me4bpy, (MeO)2bpy, (EtO)2bpy, Cl2bpy, and NO2phen, have been measured. The acid dissociation constants of ground and excited states have been determined. The ground-state pKa values were obtained from the pH dependence of the complex absorbance changes. The excited-state pKa* values were extracted from the emission titration curve and corrected for the excited-state lifetime of both protonated and deprotonated species. The largest DeltapKa, pKa*-pKa, found for Ru(Me2bpy)2(MebpyCOOH)2+ and Ru(Me4bpy)2(MebpyCOOH)2+ of 1.7 indicate that MebpyCOOH gains most of the MLCT excited-state electron. The big negative DeltapKa found for Ru(Cl2bpy)2(MebpyCOOH)2+, -4.2, clearly indicates the metal-to-ligand charge transfer to the Cl2bpy ligands.     

Inhibition of thioredoxin and thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in vivo

Lipid peroxidation is a cellular process that takes place under physiological conditions and particularly after oxidative stress. 4-Hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, is known to exert a multitude of biological effects and has high reactivity to various cellular components, including DNA and protein. The thioredoxin system, composed of the selenoenzyme thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, plays a key role in redox regulation and is involved in many signaling pathways. The selenocysteine (Sec) and cysteine (Cys) residues (Cys-496/Sec-497) in the active site of TrxR and a pair of Cys residues (Cys-32/Cys-35) in Trx are sensitive to various alkylating reagents. Herein, we report a mechanistic study on the inhibition of rat TrxR by HNE. The inhibition occurs with TrxR only in its reduced form and persists after removal of HNE. Inhibition of TrxR by HNE added to cultured HeLa cells is also observed. In addition, HNE inactivates reduced Escherichia coli Trx irreversibly. We proved that the redox residues (Cys-496/Sec-497 in TrxR and Cys-32/Cys-35 in Trx) were primary targets for HNE modification. The covalent adducts formed between HNE and Trx were also confirmed by mass spectrum. Because the thioredoxin system is one of the core regulation enzymes of cells' function, inhibition of both TrxR and Trx by HNE provides a possibly novel mechanism for explanation of its cytotoxic effect and signaling activity, as well as the further damage indirectly caused under oxidative stress conditions.     

Sulfur K-edge spectroscopic investigation of second coordination sphere effects in oxomolybdenum-thiolates: relationship to molybdenum-cysteine covalency and electron transfer in sulfite oxidase

Second-coordination sphere effects such as hydrogen bonding and steric constraints that provide for specific geometric configurations play a critical role in tuning the electronic structure of metalloenzyme active sites and thus have a significant effect on their catalytic efficiency. Crystallographic characterization of vertebrate and plant sulfite oxidase (SO) suggests that an average O(oxo)-Mo-S(Cys)-C dihedral angle of approximately 77 degrees exists at the active site of these enzymes. This angle is slightly more acute (approximately 72 degrees) in the bacterial sulfite dehydrogenase (SDH) from Starkeya novella. Here we report the synthesis, crystallographic, and electronic structural characterization of Tp*MoO(mba) (where Tp* = (3,5-dimethyltrispyrazol-1-yl)borate; mba = 2-mercaptobenzyl alcohol), the first oxomolybdenum monothiolate to possess an O(ax)-Mo-S(thiolate)-C dihedral angle of approximately 90 degrees . Sulfur X-ray absorption spectroscopy clearly shows that O(ax)-Mo-S(thiolate)-C dihedral angles near 90 degrees effectively eliminate covalency contributions to the Mo(xy) redox orbital from the thiolate sulfur. Sulfur K-pre-edge X-ray absorption spectroscopy intensity ratios for the spin-allowed S(1s) --> Sv(p) + Mo(xy) and S(1s) --> Sv(p) + Mo(xz,yz) transitions have been calibrated by a direct comparison of theory with experiment to yield thiolate Sv(p) orbital contributions, c(j)(2), to the Mo(xy) redox orbital and the Mo(xz,yz) orbital set. Furthermore, these intensity ratios are related to a second coordination sphere structural parameter, the O(oxo)-Mo-S(thiolate)-C dihedral angle. The relationship between Mo-S(thiolate) and Mo-S(dithiolene) covalency in oxomolydenum systems is discussed, particularly with respect to electron-transfer regeneration in SO.     

Catalysis of the cleavage of uridine 3'-2,2,2-trichloroethylphosphate by a designed helix-loop-helix motif peptide

A 42-residue peptide that folds into a helix-loop-helix motif and dimerizes to form a four-helix bundle has been designed to catalyze the hydrolysis of phosphodiesters. The active site on the surface of the folded catalyst is composed of two histidine and four arginine residues, with the capacity to provide general acid, general base, and/or nucleophilic catalysis as well as transition state stabilization. Uridine 3'-2,2,2 trichloroethylphosphate (2) is a mimic of RNA with a leaving group pKa of 12.3. Its hydrolysis is energetically less favorable than that of commonly used model substrates with p-nitrophenyl leaving groups and therefore a more realistic model for the design of catalysts capable of cleaving RNA. The second-order rate constant for the hydrolysis of 2 at pH 7.0 by the polypeptide catalyst was 418 x 10(-6) M-1 s-1, and that of the imidazole catalyzed reaction was 1.66 x 10(-6) M-1 s-1. The pH dependence suggested that catalysis is due to the unprotonated form of a residue with a pKa of around 5.3, and the observed kinetic solvent isotope effect of 1.9 showed that there is significant hydrogen bonding in the transition state, consistent with general acid-base catalysis. The rate constant ratio k2(Pep)/k2(Im) of 252 is probably due to a combination of nucleophilic and general acid-base catalysis, as well as transition state stabilization. Substrate binding was weak since no sign of saturation kinetics was observed for substrate concentrations in the range from 5 to 40 mM. The results provide a platform for the further development of catalysts for RNA cleavage with a potential role in the development of drugs.     

Unexpectedly fast cis/trans isomerization of Xaa-Pro peptide bonds in disulfide-constrained cyclic peptides

Acyclic dithiol and cyclic disulfide forms of the peptides Ac-Cys-Pro-Xaa-Cys-NH2 (Xaa = Phe, His, Tyr, Gly, and Thr) and Ac-Cys-Gly-Pro-Cys-NH2 and the peptide Ac-Ala-Gly-Pro-Ala-NH2 were synthesized and characterized by mass spectrometry and NMR spectroscopy. Rate constants kct and ktc for cis-to-trans and trans-to-cis isomerization, respectively, across the Cys-Pro or Gly-Pro peptide bonds were determined by magnetization transfer NMR techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. It was found that constraints imposed by the disulfide bond confer an unexpected rate enhancement for cis/trans isomerization, ranging from a factor of 2 to 13. It is proposed that the rate enhancements are a result of an intramolecular catalysis mechanism in which the NH proton of the Pro-Xaa peptide bond hydrogen bonds to the proline nitrogen in the transition state. The peptides Ac-Cys-Pro-Xaa-Cys-NH2 and Ac-Cys-Gly-Pro-Cys-NH2 are model compounds for proline-containing active sites of the thioredoxin superfamily of oxidoreductase enzymes; the results suggest that the backbones of the active sites of the oxidized form of these enzymes may have unusual conformational flexibility.