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In this review, we summarize the advance of stimuli-responsive pillararene modified nanosystems for controlled release of drugs from the perspectives of decomposition release and gated release, and describe in detail the controlled release of recently developed photo-, pH-, thermal-, chemical- and multi-responsive nanosystems.

The rapid extraction of high-purity nucleic acids from complex biological samples using conventional methods is complicated. Therefore, in this study, glycine-pillar[6]arene (Gly-P6)-functionalized tapered nanochannels were constructed using 32-mer single-stranded

The extraction efficiency, recovery, quantification, and stability of isoflavones (daidzein, genistein and puerarin) from the wastes /by-products of kudzu roots and soy molasses were evaluated by using natural deep eutectic solvents (NADESs) coupled with ultrasound-assisted extraction. The NADES extraction was compared with the conventional Soxhlet extraction technique, and the extracted isoflavones were quantified using HPLC-UV/Vis. Results showed extractability of puerarin to be significantly increased (p < 0.0001) in proportion to the increased water concentration of NADES (9.7 ± 0.2, 12.6 ± 0.2, and 14.8 ± 0.3 mg/g in NADESs containing 10%, 20%, and 30% water, respectively). Despite having less isoflavone content, kudzu roots and soy molasses extracts obtained using NADES exhibited higher antioxidant activity when compared with methanolic extracts. Further, NADES extracts also markedly reduced the degradation degree of isoflavones. The results of this investigation support the use of green extraction techniques to obtain value-added components like isoflavones from wastes/by-products like that of kudzu roots and soy molasses, which are envisaged to find future potential applications in the food and pharmaceutical industries. © 2022 Elsevier B.V. Russian Science Foundation, RSF: 20–66-47017 This work was supported by the Russian Science Foundation RSF (Grant agreement number 20–66-47017 ). The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Elena G. Kovaleva reports financial support was provided by Russian Science Foundation RSF (Grant agreement number 20–66–47017).

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75Å resolution structure of the H200Q variant was determined from ∼16min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the second-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here, the high-spin Fe(II) states of resting H200C and the H200C-homoprotocatechuate enzyme-substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin Fe(II) center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ - 8 cm(-1), E/D ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the (57)Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 Fe(II) system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation, which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well.

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mossbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mossbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.

Statement of the Problem: Many solid-phase materials, for instance, porous and nano structural objects as well as the systems of specific functionality are widely used in aqueous solutions as promising heterogeneous catalysts of various reactions, can serve as suitable carriers for catalytically active organic and bioorganic groups and enzymes, and also be adsorbents of large and small molecules. The properties of solid-phase materials are affected therefore, both the chemical nature of the solution and some specific conditions are arising in the phase and on the surface of these materials. Surface electrical potential (SEP) is among the most important surface characteristics of these materials. At present, there is no method for measuring SEP of hydrated porous and nanostructured materials. Some of the most important parameters of mesoporous materials are Stem potential of double electrical layer (DEL) and surface charge because of the pivotal role they play in the adsorption of charged molecular species. There are not any conventional and easy-to-perform techniques used to measure Stern potential in porous systems. The purpose of this work is to measure Stern potential in a specific case, i.e., practically cylindrical nanosized hydrated channels of the mesoporous molecular sieves (MMS) MCM-41 and SBA-15 with channel diameters ranging from 2.3 to 8.1 nm using pH-sensitive nitroxide radicals (NR). A technique for direct measurement of the near-surface (Stern) potential using pH-sensitive NR was developed. Negative and positive values of Stern potential for the positively and negatively charged surface of MMS channels were found. The ranges of pH of external solution for the near-zero charge, boost-charging of the channel surface and for dissociation of functional groups in the MMS studied were found. Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. Aim: The aim of this study is to develop the method for measuring SEP of different solid phase hydrated materials by EPR of pH sensitive nitroxides (NR) as spin probes and labels. Methodology & Theoretical Orientation: A variety of nitroxides with the range of pH-sensitivity from 2 to 8.5 pH were incorporated into nanoporous and nanostructured inorganic oxides as powders and membranes and a diversity of organoinorganic hybride materials both by adsorption from aqueous solution and through covalent binding technique. pH-dependent parameters of EPR spectra were measured through monitoring pH in a bulk solution and inside materials. Anionic phospholipids play many roles in biophysical processes. Findings: The negative and positive values of stern potential for the positively and negatively charged surfaces were measured from the characteristic shifts of the EPR titration curves for the slow-motion NR located in the material near-surface about those for a bulk solution. Stern layer thickness for mesoporous silicas was determined from the near-surface electrical potential profile using a model of practically cylindrical nano sized hydrated channels of the mesoporous silicas with channel diameters ranging from 2.3 to 8.1 nm. Conclusion & Significance: An unique technique for measuring the near-surface (Stern) potential as well as Stern layer thickness and surface charge based on EPR of pH-sensitive nitroxides as spin probes and labels have been developed for a wide range of hydrated porous and nanostructured materials with a great potential for adsorption processes and heterogeneous catalysis.

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). Whereas Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently, exhibiting significantly higher $$ K_{\text{M}}^{{{\text{O}}_{ 2} }} $$ and k cat. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-HPCD, Mn-HPCD, and Co-HPCD shows that metal substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady-state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe-HPCD and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O2 to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O2 activation mechanism associated with the extradiol dioxygenase family.

The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. Sequences of naturally biotinylated substrates are highly conserved across evolutionary boundaries, and cross-species biotinylation has been demonstrated in several systems. To define the minimal substrate requirements in BirA-catalyzed biotinylation, we have measured the kinetics of modification of a 23-residue peptide previously identified by combinatorial methods. Although the sequence of the peptide bears little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo. Rates of biotin transfer to the 23-residue peptide are similar to those determined for BCCP. To further elucidate the sequence requirements for biotinylation, transient kinetic measurements were performed on a series of amino- and carboxy-terminal truncations of the 23-mer. The results, determined by stopped-flow fluorescence, allowed identification of a 14-residue peptide as the minimum required sequence. Additional support was obtained using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides that had been incubated with an excess of biotinyl-5'-adenylate intermediate and catalytic amounts of BirA. Results of these measurements indicate that while kinetically inactive truncations showed no significant shift in molecular mass to the values expected for biotinylated species, kinetically active truncations exhibited 100% biotinylation. The specificity constant (k(cat)/Km) governing BirA-catalyzed biotinylation of the 14-mer minimal substrate is similar to that determined for the natural substrate, BCCP. We conclude that the 14-mer peptide efficiently mimics the biotin acceptor function of the much larger protein domain normally recognized by BirA.

Biological O 2 activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-to-O 2 electron transfer results in a reactive M(III)-superoxo species. For the extradiol aromatic ring-cleaving dioxygenases, we have proposed a different model where an electron is transferred from substrate to O 2 via the M(II) center to which they are both bound, thereby obviating the need for an integral change in metal redox state. This model is tested by using homoprotocatechuate 2,3-dioxygenases from Brevibacterium fuscum (Fe-HPCD) and Arthrobacter globiformis (Mn-MndD) that share high sequence identity and very similar structures. Despite these similarities, Fe-HPCD binds Fe(II) whereas Mn-MndD incorporates Mn(II). Methods are described to incorporate the nonphysiological metal into each enzyme (Mn-HPCD and Fe-MndD). The x-ray crystal structure of Mn-HPCD at 1.7 Å is found to be indistinguishable from that of Fe-HPCD, while EPR studies show that the Mn(II) sites of Mn-MndD and Mn-HPCD, and the Fe(II) sites of the NO complexes of Fe-HPCD and Fe-MndD, are very similar. The uniform metal site structures of these enzymes suggest that extradiol dioxygenases cannot differentially compensate for the 0.7-V gap in the redox potentials of free iron and manganese. Nonetheless, all four enzymes exhibit nearly the same K M and V max values. These enzymes constitute an unusual pair of metallo-oxygenases that remain fully active after a metal swap, implicating a different way by which metals are used to promote oxygen activation without an integral change in metal redox state.

Oxidase and oxygenase enzymes allow the use of relatively unreactive O2 in biochemical reactions. Many of the mechanistic strategies employed in nature for this key reaction are represented within the 2-His-1-carboxylate facial triad family of non-heme Fe(II) containing enzymes. The open face of the metal coordination sphere opposite the three endogenous ligands participates directly in the reaction chemistry. Here, data from several studies are presented showing that reductive O2 activation within this family is initiated by substrate (and in some cases co-substrate or cofactor) binding, which then allows coordination of O2 to the metal. From this starting point, both the O2 activation process and the reactions with substrates diverge broadly. The reactive species formed in these reactions have been proposed to encompass four oxidation states of iron and all forms of reduced O2 as well as several of the reactive oxygen species that derive from O–O bond cleavage.

Intermediates in the reaction cycle of an oxygenase are usually very informative with respect to the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.

Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species.

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-A resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-550 s(-1)) is a common step that precedes binding of all inhibitors. At all pH values studied, the rate constants for proton release or uptake matched those for formation of ternary complexes, and the most significant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) was observed for enzyme species involved in deprotonation steps. Kinetic simulations of the combined transient data for the multiple signals indicate that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD+ complex, which has a pK of about 7.3. The results suggest that rate-limiting deprotonation of the enzyme-NAD+ complex is coupled to the conformational change and controls the formation of ternary complexes.

The reactive oxy intermediate of the catalytic cycle of extradiol aromatic ring-cleaving dioxygenases is formed by binding the catecholic substrate and O2 in adjacent ligand positions of the active site metal [usually Fe(II)]. This intermediate and the following Fe(II)-alkylperoxo intermediate resulting from oxygen attack on the substrate have been previously characterized in a crystal of homoprotocatechuate 2,3-dioxygenase (HPCD). Here a subsequent intermediate in which the O-O bond is broken to yield a gem diol species is structurally characterized. This new intermediate is stabilized in the crystal by using the alternative substrate, 4-sulfonylcatechol, and the Glu323Leu variant of HPCD, which alters the crystal packing.

Homoprotocatechuate 2,3-dioxygenase (FeHPCD) utilizes an active site FeII to activate O2 in a reaction cycle that ultimately results in aromatic ring cleavage. Here, the roles of the conserved active site residue Tyr257 are investigated by solving the X-ray crystal structures of the Tyr257-to-Phe variant (Y257F) in complex with the substrate homoprotocatechuate (HPCA) and the alternative substrate 4-nitrocatechol (4NC). These are compared with structures of the analogous wild type enzyme complexes. In addition, the oxy intermediate of the reaction cycle of Y257F-4NC + O2 is formed in crystallo and structurally characterized. It is shown that both substrates adopt a previously unrecognized, slightly non-planar, strained conformation affecting the geometries of all aromatic ring carbons when bound in the FeHPCD active site. This global deviation from planarity is not observed for the Y257F variant. In the Y257F-4NC-oxy complex, the O2 is bound side-on to the FeII, while the 4NC is chelated in two adjacent sites. The ring of the 4NC in this complex is planar, in contrast to the equivalent FeHPCD intermediate, which exhibits substantial local distortion of the substrate hydroxyl moiety (C2-O−) that is hydrogen bonded to Tyr257. We propose that Tyr257 induces the global and local distortions of the substrate ring in two different ways. First, van der Waals conflict between the Tyr257-OH substituent and the substrate C2 carbon is relieved by adopting the globally strained structure. Second, Tyr257 stabilizes the localized out-of-plane position of the C2-O− by forming a stronger hydrogen bond as the distortion increases. Both types of distortion favor transfer of one electron out of the substrate to form a reactive semiquinone radical. Then, the localized distortion at substrate C2 promotes formation of the key alkylperoxo intermediate of the cycle resulting from oxygen attack on the activated substrate at C2, which becomes sp3 hybridized. The inability of Y257F to promote the distorted substrate structure may explain the observed 100-fold decrease in the rates of the O2 activation and insertion steps of the reaction. Homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum catalyzes the fission of the O-O bond of O2 with incorporation of both atoms into the aromatic substrate (HPCA) resulting in ring cleavage as shown in Scheme 1.1–5 The mechanism shown in Scheme 1 begins with O2 and HPCA binding in adjacent coordination sites of the active site FeII.4,6–10 Electron transfer from HPCA to the O2 via the iron would give both substrates radical character, allowing facile recombination to form an alkylperoxo intermediate from which O-O bond fission and aromatic ring cleavage could ensue. Both the initial intermediate, in which each substrate has radical character, and the alkylperoxo intermediate resulting from oxygen attack have been structurally characterized by X-ray crystallography after a crystal of the enzyme soaked with the slow substrate 4-nitrocatechol (4NC) was exposed to low concentrations of O2. 3 While the chemical nature of the alkylperoxo species is apparent from the structure, the diradical character of the initial intermediate was surmised from the pronounced lack of planarity observed for the aromatic substrate. It was proposed that the non-planar ring results from a localized semiquinone radical (SQ•) on the ring C2 (See Scheme 1 for numbering system used here) where the attack by oxygen (at the level of O2•−) will occur. Accordingly, in the alkylperoxo intermediate, this carbon in observed to be fully sp3 hybridized and 4 coordinate. Scheme 1 Proposed reaction mechanism for extradiol dioxygenases. In the case of FeHPCD, R is –CH2COO− (optimal substrate HPCA) or –NO2 (alternative substrate 4NC), B(H) is His200. The ring carbon numbering system shown is adopted for both ... The structural studies from our laboratory and others have shown that there are several second sphere active site residues that may contribute to catalysis (e.g. Figure 1).3,5,9–11 Extensive investigations of an active site His (His200 in the case of FeHPCD) have shown that it plays many roles including stabilization of the Fe-bound O2 via hydrogen bonding and charge interaction, acid-base catalysis of the alkylperoxo intermediate formation and subsequent oxygen insertion chemistry, and possibly, stabilization of the bound oxygen in the side-on orientation properly aligned to attack the aromatic substrate.3,12–15 Intermediates trapped from solution reactions of FeHPCD and its variants at His200 show that iron converts transiently to the FeIII state in the absence of H200, but it is always observed in the FeII state in its presence. 13,14 Thus, when His200 is present, one or two electrons appear to pass from the aromatic substrate to the oxygen with either no change in iron oxidation state or a transient change that persists for much less than the freezing time for rapid freeze quench (RFQ) experiments (~10 ms).14 This suggests that there is a considerable driving force for electron transfer that is not apparent from the redox potentials of the isolated aromatic substrate and O2. Figure 1 Active site environment of FeHPCD in complex with the optimal substrate HPCA (PDB 4GHG, subunit C). Atom color code: gray, carbon (enzyme); yellow, carbon (substrate); blue, nitrogen; red, oxygen; bronze, iron. Red dashed lines show hydrogen bonds (A). ... Another second sphere amino acid, Tyr257, is hydrogen bonded to the deprotonated C2-O− of bound HPCA (Figure 1). The orientation of Tyr257 is such that this interaction might stabilize the non-planar structure of the substrate in the reaction cycle intermediate. In the accompanying report,16 it is shown that mutation of Tyr257 to Phe (Y257F) results in a significant decrease in the rate constants for many steps with reduction approaching 100-fold in the oxygen binding and insertion steps despite the presence of the acid-base catalyst His200. The results suggest that the putative HPCA SQ•-FeII-O2•− is either not formed or is unreactive, leading to formation of an observed HPCA quinone-FeII-(H)peroxo species that then slowly yields the correct ring cleaved product. Here, structural studies of the Y257F variant, its complexes with HPCA and 4NC, and the intermediate that results from exposure of the Y257F-4NC complex to O2 are reported. The results give insight into the structural basis for the observed decreases in rate constants and the change in the chemical nature of the reactive intermediate as well as the strategy used by aromatic dioxygenases to simultaneously maintain high rates and high fidelity in ring cleaving reactions.

We report the structures of three intermediates in the O 2 activation and insertion reactions of an extradiol ring-cleaving dioxygenase. A crystal of Fe 2+ -containing homoprotocatechuate 2,3-dioxygenase was soaked in the slow substrate 4-nitrocatechol in a low O 2 atmosphere. The x-ray crystal structure shows that three different intermediates reside in different subunits of a single homotetrameric enzyme molecule. One of these is the key substrate-alkylperoxo-Fe 2+ intermediate, which has been predicted, but not structurally characterized, in an oxygenase. The intermediates define the major chemical steps of the dioxygenase mechanism and point to a general mechanistic strategy for the diverse 2-His-1-carboxylate enzyme family.

X-ray exposure during crystallographic data collection can result in unintended redox changes in proteins containing functionally important redox centers. In order to directly monitor X-ray-derived redox changes in trapped oxidative half-reaction intermediates of Paracoccus denitrificans methylamine dehydrogenase, a commercially available single-crystal UV/Vis microspectrophotometer was installed on-line at the BioCARS beamline 14-BM-C at the Advanced Photon Source, Argonne, USA. Monitoring the redox state of the intermediates during X-ray exposure permitted the creation of a general multi-crystal data collection strategy to generate true structures of each redox intermediate.

S-(N-Aryl-N-hydroxycarbamoyl)glutathione derivatives (GSC(O)N(OH)C6H4X, where GS = glutathionyl and X = H (1), Cl (2), Br (3)) have been proposed as possible anticancer agents, because of their ability to strongly inhibit the methylglyoxal-detoxifying enzyme glyoxalase I. In order to test this hypothesis, the in vitro antitumor activities of these compounds and their [glycyl,glutamyl] diethyl ester prodrug forms (1(Et)2-3(Et)2) have been examined. All three diethyl esters inhibit the growth of L1210 murine leukemia and B16 melanotic melanoma in culture, with GI50 values in the micromolar concentration range. Cell permeability studies with L1210 cells indicate that growth inhibition is associated with rapid diffusion of the diethyl esters into the cells, followed by enzymatic hydrolysis of the ethyl ester functions to give the inhibitory diacids. In contrast, the corresponding diacids neither readily diffuse into nor significantly inhibit the growth of these cells. Consistent with the hypothesis that cell growth inhibition is due to competitive inhibition of glyoxalase I, preincubation of L1210 cells with 2(Et)2 increases the sensitivity of these cells to the inhibitory effects of exogenous methylglyoxal. Compound 2(Et)2 is much less toxic to nonproliferating murine splenic lymphocytes, possibly reflecting reduced sensitivity to methylglyoxal and/or reduced chemical stability of the diacid inside these cells. Finally, a plasma esterase-deficient murine model has been identified that should allow in vivo testing of the diethyl esters.