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Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA-repairing photolyases. The animal-like cryptochrome CraCRY from the green alga Chlamydomonas reinhardtii challenges the paradigm of cryptochromes as pure blue-light receptors by acting as a (6-4) photolyase, using 8-hydroxy-5-deazaflavin (8-HDF) as a light-harvesting antenna with a 17.4 Å distance to flavin and showing spectral sensitivity up to 680 nm. The expanded action spectrum is attributed to the presence of the flavin neutral radical (FADH•) in the dark, despite a rapid FADH• decay observed in vitro in samples exclusively carrying flavin. Herein, the red-light response of CraCRY carrying flavin and 8-HDF was studied, revealing a 3-fold prolongation of the FADH• lifetime in the presence of 8-HDF. Millisecond time-resolved ultraviolet-visible spectroscopy showed the red-light-induced formation and decay of an absorbance band at 458 nm concomitant with flavin reduction. Time-resolved Fourier transform infrared (FTIR) spectroscopy and density functional theory attributed these changes to the deprotonation of 8-HDF, challenging the paradigm of 8-HDF being permanently deprotonated in photolyases. FTIR spectra showed changes in the hydrogen bonding network of asparagine 395, a residue suggested to indirectly control flavin protonation, indicating the involvement of N395 in the stabilization of FADH•. Fluorescence spectroscopy revealed a decrease in the energy transfer efficiency of 8-HDF upon flavin reduction, possibly linked to 8-HDF deprotonation. The discovery of the interdependence of flavin and 8-HDF beyond energy transfer processes highlights the essential role of the antenna, introducing a new concept enabling CraCRY and possibly other bifunctional cryptochromes to fulfill their dual function.

In a continuing effort to identify structural attributes required for strong binding and potent inhibition of human drug-metabolizing CYP3A4, we designed ten ritonavir-like analogues differing in the side-group stereochemistry, backbone atomic composition, and headgroup spacing. All analogues had pyridine and tert-butyloxycarbonyl (Boc) as the heme-ligating head and tail groups, respectively, phenyl side groups, and either a methyl- or ethyl-pyridyl linker. Each linker subseries had S/ R, R/ S, R/ R, and S/S side-group conformers (4a-d and 4e-h, respectively), and one S/S stereoisomer with the backbone S-to-N-heteroatom substitution (6a and 6b). To elucidate structure-activity relationships, ligand-dependent changes in optical spectra, dissociation constant ( Ks), inhibitory potency (IC50), thermostability, and heme ligation and reduction kinetics were analyzed. Comparison of the subseries and individual compounds showed that CYP3A4 only weakly discriminates between side-group configurations, associates more tightly with the pyridyl-ethyl-linker analogues, and strongly disfavors the N-containing backbone. Ks and IC50 for the pyridyl-ethyl R/ R conformer, 4g, were the lowest and close to those for ritonavir: 0.04 and 0.31 μM versus 0.02 and 0.13 μM, respectively. Determination of the X-ray structures of the inhibitory complexes was critical for experimental data interpretation, especially for the uniquely oriented 4a and 4e. Based on structural analysis, we conclude that, for this series of analogues, the ligand-mediated interactions near the heme are dominant and define the binding mode and that fine-tuning of these interactions as well as the backbone spacing could further improve the affinity and inhibitory strength.

It is now recognized that many amyloid-forming proteins can associate into multiple fibril structures. Here, we use two-dimensional infrared spectroscopy to study two fibril polymorphs formed by human islet amyloid polypeptide (hIAPP or amylin), which is associated with type 2 diabetes. The polymorphs exhibit different degrees of structural organization near the loop region of hIAPP fibrils. The relative populations of these polymorphs are systematically altered by the presence of macrocyclic peptides which template β-sheet formation at specific sections of the hIAPP sequence. These experiments are consistent with polymorphs that result from competing pathways for fibril formation and that the macrocycles bias hIAPP aggregation toward one pathway or the other. Another macrocyclic peptide that matches the loop region but extends the lag time leaves the relative populations of the polymorphs unaltered, suggesting that the branching point for structural divergence occurs after the lag phase, when the oligomers convert into seeds that template fibril formation. Thus, we conclude that the structures of the polymorphs stem from restricting oligomers along diverging folding pathways, which has implications for drug inhibition, cytotoxicity, and the free energy landscape of hIAPP aggregation.

The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron-sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis. We have further examined the differences between these two P450s by determining the effect of spin equilibrium, redox properties, and stability of oxygen complexes. We find that Arx shifts the spin state equilibrium toward high-spin, which is the opposite of the effect of Pdx on P450cam. In both P450s, redox partner binding destabilizes the oxy-P450 complex but this effect is much weaker with CYP101D1. In addition, resonance Raman data show that structural perturbations observed in P450cam upon addition of Pdx are absent in CYP101D1. These data indicate that Arx does not play the same effector role in catalysis as Pdx does with P450cam. The most relevant structural difference between these two P450s centers on a catalytically important Asp residue required for proton-coupled electron transfer. We postulate that with P450cam larger Pdx-assisted motions are required to free this Asp for catalysis while the smaller number of restrictions in CYP101D1 precludes the need for redox partner-assisted structural changes.

Dopa (l-3,4-dihydroxyphenylalanine) is a key chemical signature of mussel adhesive proteins, but its susceptibility to oxidation has limited mechanistic investigations as well as practical translation to wet adhesion technology. To investigate peptidyl-Dopa oxidation, the highly diverse chemical environment of Dopa in mussel adhesive proteins was simplified to a peptidyl-Dopa analogue, N-acetyl-Dopa ethyl ester. On the basis of cyclic voltammetry and UV-vis spectroscopy, the Dopa oxidation product at neutral to alkaline pH was shown to be α,β-dehydro-Dopa (ΔD), a vinylcatecholic tautomer of Dopa-quinone. ΔD exhibited an adsorption capacity on TiO2 20-fold higher than that of the Dopa homologue in the quartz crystal microbalance. Cyclic voltammetry confirmed the spontaneity of ΔD formation in mussel foot protein 3F at neutral pH that is coupled to a change in protein secondary structure from random coil to β-sheet. A more complete characterization of ΔD reactivity adds a significant new perspective to mussel adhesive chemistry and the design of synthetic bioinspired adhesives.

Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msmeg) can grow on cholesterol as the sole carbon source. In Mtb the utilization of cholesterol can be initiated by CYP125A1 or CYP142A1 and in Msmeg by the orthologous CYP125A3 and CYP142A2. Double knockout of the two enzymes in Mtb prevents its growth on cholesterol, but the double knockout of Msmeg is still able to grow, albeit at a slower rate. We report here that Msmeg has a third enzyme, CYP125A4, that also oxidizes cholesterol, although it has a much higher activity for the oxidation of 7α-hydroxycholesterol. The ability of Msmeg CYP125A4 (and Mtb CYP125A1) to oxidize 7α-hydroxycholesterol is due, at least in part, to the presence of a smaller amino acid side chain facing C-7 of the sterol substrate than in CYP125A3. The ability to oxidize 7-substituted steroids broadens the range of sterol carbon sources for growth, but even more importantly in Mtb, additional biological effects are possible due to the potent immunomodulatory activity of 7α,26-dihydroxycholesterol.

Evidence of a CO docking site near the FeMo cofactor in nitrogenase has been obtained by Fourier transform infrared spectroscopy-monitored low-temperature photolysis. We investigated the possible migration paths for CO from this docking site using molecular dynamics calculations. The simulations support the notion of a gas channel with multiple internal pockets from the active site to the protein exterior. Travel between pockets is gated by the motion of protein residues. Implications for the mechanism of nitrogenase reactions with CO and N2 are discussed.

HydE and HydG are radical S-adenosyl-l-methionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the nonprotein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxides, three cyanides, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatic, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-l-methionine. Incorporation of deuterium from D2O into 5'-deoxyadenosine, the cleavage product of S-adenosyl-l-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, has allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster is likely derived from HydE's thiol containing substrate.

A high-throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes for modulating gene regulation mediated by VDR. Peroxisome proliferator-activated receptor (PPAR) δ agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations of > 12.1 μM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor. Surprisingly, GW0742 behaved as a PPAR agonist and antagonist, activating transcription at lower concentrations and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742 increased the fluorescence intensity and level of fluorescence polarization at an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a dose-dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced level of expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3, and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.

Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoenzyme that catalyzes the oxidation of d-glucose and other aldopyranose sugars at the C2 position by using O2 as an electron acceptor to form the corresponding 2-keto-sugars and H2O2. In this study, the effects of pH on the oxidative half-reaction of P2O were investigated using stopped-flow spectrophotometry. The results showed that flavin oxidation occurred via different pathways depending on the pH of the environment. At pH values lower than 8.0, reduced P2O reacts with O2 to form a C4a-hydroperoxyflavin intermediate, leading to elimination of H2O2. At pH 8.0 and higher, the majority of the reduced P2O reacts with O2 via a pathway that does not allow detection of the C4a-hydroperoxyflavin, and flavin oxidation occurs with decreased rate constants upon the rise in pH. The switching between the two modes of P2O oxidation is controlled by protonation of a group which has a pKa of 7.6 ± 0.1. Oxidation reactions of reduced P2O under rapid pH change as performed by stopped-flow mixing were different from the same reactions performed with enzyme pre-equilibrated at the same specified pH values, implying that the protonation of the group which controls the mode of flavin oxidation cannot be rapidly equilibrated with outside solvent. Using a double-mixing stopped-flow experiment, a rate constant for proton dissociation from the reaction site was determined to be 21.0 ± 0.4 s-1. [ABSTRACT FROM AUTHOR]

This work examines the effect of perturbing the position of bound CO2 in the active site of human carbonic anhydrase II (HCA II) on catalysis. Variants of HCA II in which Val143 was replaced with hydrophobic residues Ile, Leu, and Ala were examined. The efficiency of catalysis in the hydration of CO2 for these variants was characterized by 18O exchange mass spectrometry, and their structures were determined by X-ray crystallography at 1.7-1.5 Å resolution. The most hydrophobic substitutions, V143I and V143L, showed decreases in the level of catalysis, as much as 20-fold, while the replacement by the smaller V143A mutation showed an only moderate 2-fold decrease in activity. Structural data for all three variants show no significant change in the overall position of amino acid side chains in the active site compared with the wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to the number for the wild type. To further investigate the decrease in the catalytic efficiency of V143I HCA II, an X-ray crystallographic CO2 entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift in the CO2 substrate toward the zinc-bound solvent, placing it ∼0.3 Å closer than previously observed in the wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the acquisition of data. These data suggest that the Ile substitution at position 143 reduced the catalytic efficiency, which is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO2 into bicarbonate and a decreased product dissociation rate. [ABSTRACT FROM AUTHOR]

Low-density lipoprotein (LDL) has recently been shown to be oxidized by iron within the lysosomes of macrophages, and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterize the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α- tocopherol on this process. LDL was oxidized by iron at pH 4.5 and 37 °C and its oxidation monitored by spectrophotometry and high-performance liquid chromatography. LDL was oxidized effectively by FeSO4 (5-50 μM) and became highly aggregated at pH 4.5, but not at pH 7.4. The level of cholesteryl esters decreased, and after a pronounced lag, the level of 7-ketocholesterol increased greatly. The total level of hydroperoxides (measured by the triiodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.3 and 37 °C was similar to that of LDL oxidized by copper at pH 7.4 and 4 °C, i.e., rich in hydroperoxides but low in oxysterols. Previously oxidized LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous iron was much more effective than ferric iron at oxidizing LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepenta-acetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages but were unable to prevent aggregation of LDL after it had been partially oxidized. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the rate of oxidations of LDL but decreased it later. The presence of oxidized and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis. [ABSTRACT FROM AUTHOR]

The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188Ser residue substituted by cysteine and the other having the β-153Cys residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s-1) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s-1) from the Fe protein [4Fe -4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β- 153Cys deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ- methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF4- as a posthydrolysis analogue (a mimic of ADP + Pi). [ABSTRACT FROM AUTHOR]

Expression of six genes from two glutamate fermenting clostridia converted Escherichia coli into a producer of glutaconate from 2-oxoglutarate of the general metabolism (Djurdjevic, I. et al. 2010, Appl. Environ. Microbiol.77, 320-322). The present work examines whether this pathway can also be used to reduce 2-oxoadipate to (R)-2-hydroxyadipic acid and dehydrate its CoA thioester to 2-hexenedioic acid, an unsaturated precursor of the biotechnologically valuable adipic acid (hexanedioic acid). 2-Hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum, the key enzyme of this pathway and a potential radical enzyme, catalyzes the reversible dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA. Using a spectrophotometric assay and mass spectrometry, it was found that (R)-2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA, muconyl-CoA, and butynedioyl-CoA, but not 3-methylglutaconyl-CoA, served as alternative substrates. Hydration of butynedioyl-CoA most likely led to 2-oxosuccinyl-CoA, which spontaneously hydrolyzed to oxaloacetate and CoASH. The dehydratase is not specific for the CoA-moiety because (R)-2-hydroxyglutaryl-thioesters of N-acetylcysteamine and pantetheine served as almost equal substrates. Whereas the related 2-hydroxyisocaproyl-CoA dehydratase generated the stable and inhibitory 2,4-pentadienoyl-CoA radical, the analogous allylic ketyl radical could not be detected with muconyl-CoA and 2-hydroxyglutaryl-CoA dehydratase. With the exception of (R)-2-hydroxyglutaryl-CoA, all mono-CoA-thioesters of dicarboxylates used in this study were synthesized with glutaconate CoA-transferase from Acidaminococcus fermentans. The now possible conversion of (R)-2-hydroxyadipate via (R)-2-hydroxyadipoyl-CoA and 2-hexenedioyl-CoA to 2-hexenedioate paves the road for a bio-based production of adipic acid. [ABSTRACT FROM AUTHOR]

Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and iow turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased aflinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNRincreased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimeraemulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes intheir overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency. of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs. [ABSTRACT FROM AUTHOR]

PQQ is an exogenous, tricyclic, quino-cofactor for a number of bacterial dehydrogenases. The final step of PQQ formation is catalyzed by PqqC, a cofactorless oxidase. This study focuses on the activation of molecular oxygen in an enzyme active site without metal or cofactor and has identified a specific oxygen binding and activating pocket in PqqC. The active site variants H154N, Y175F.S, and R179S were studied with the goal of defining the site of O2 binding and activation. Using apo-glucose dehydrogenase to assay for PQQ production. none of the mutants in this "O2 core" are capable of PQQ/PQQH2 formation. Spectrophotometric assays give insight into the incomplete reactions being catalyzed by these mutants. Active site variants Y175F, H154N, and R179S form a quinoid intermediate (Figure I) anaerobically. Y175S is capable of proceeding further from quinoid to quinol, whereas Y175F, H154N, and R179S require O2 to produce the quinol species. None of the mutations precludes substrate/product binding or oxygen binding. Assays for the oxidation of PQQH, to PQQ show that these O2 core mutants are incapable of catalyzing a rate increase over the reaction in buffer, whereas H154N can catalyze the oxidation of PQQH2 to PQQ in the presence of H2O2 as an electron acceptor. Taken together. these data indicate that none of the targeted mutants can react fully to form quinone even in the presence of bound O2. The data indicate a successful separation of oxidative chemistry from O2 binding. The residues H154, Y175. and R179 are proposed to form a core O2 binding structure that is essential for efficient O2 activation. [ABSTRACT FROM AUTHOR]

Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation events between remote sites that is integral to many biological processes, including allostery. Here we describe how the Structures and functions of two normally unlinked proteins, namely, the heme binding capability of cytochrome h562 and the antibiotic degrading β-lactamase activity of TEM, have been coupled using a directed evolution domain insertion approach. The important small biomolecule heme directly modulates in vivo and in vitro the β-lactamase activity of selected integral fusion proteins. The presence of heme decreased the concentration of ampicillin tolerated by Escherichia coli and the level of in vitro hydrolysis of nitrocefin by up to 2 orders of magnitude. Variants with the largest switching magnitudes contained insertions at second-shell sites that abut key catalytic residues. Spectrophotometry confirmed that heme bound to the integral fusion proteins in a manner similar to that of cytochrome b562. Circular dichroism suggested that only subtle structural changes rather than gross folding-unfolding events were responsible for modulating β-lactamase activity, and size exclusion chromatography confirmed that the integral fusion proteins remained monomeric in both the apo and holo forms. Thus, by sampling a variety of insertion positions and linker sequences, we are able to couple the functions of two unrelated proteins by domain insertion. [ABSTRACT FROM AUTHOR]

The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate β-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium L-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5'-phosphate and oxaloacetate or pyruvate. After consumption of the L-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate, in contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-β-hydroxy-DL-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of ∼1 or 0.006s-1, respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of L-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. it is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5'-phosphate Schiff base and increases the pKa, resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM L-Asp also results in the formation of the 325 rim complex, but with an apparent rate constant of 0.2 s-1, at least 5000-fold slower than the rate of wild-type ABDC. in contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role in modulating the pK, of the pyridoxa! 5'-phosphate complexes during catalysis. [ABSTRACT FROM AUTHOR]

Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV). whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555. 54-59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392): this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the "solution potential" does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site. [ABSTRACT FROM AUTHOR]

HPr is a central protein of the phosphoenolpyruvate:sugar phosphotrarisferase transport system (PTS). In streptococci, HPr can be phosphorylated at His15 at the expense of PEP by enzyme I (EI)of the PTS, producing HPr(His∼P). HPr can also be phosphorylated at Ser46 by the ATP-dependent HPr(Ser) kinase/ phosphorylase (HprK/P), producing HPr(Ser-P). Lastly, HPr can be phosphorylated on both residues, producing HPr(Ser-P)(His∼P) (HPr-P2). We report here a study on the phosphorylation of Streptococcus salivarius HPr, HPr(HI5D), HPr(HI5E), and HPr(His∼P) by HprK/P to assess the involvement of HprK/P in the synthesis of HPr-P2 in streptococcal cells. We first developed a spectrophotometric method for measuring HprK/P kinase activity. Using this assay, we found that the Km of HprK/P for HPr at pH 7.4 and 37 °C was approximately 110 μM, with a specificity constant (kcat/Km) of 1.7 x 104 M-1s-1. The specificity constants for HPr(HI5D) and HPr(HI5E) were 13 times lower. Kinetic studies conducted under conditions where HPr(His∼P) was stable (i.e., pH 8.6 and 15 °C) showed that HPr(His∼P) was a poorer substrate for HprK/P than HPr(HI5D), the kcat/Km for HPr(HI5D) and HPr(His∼P) being approximately 9 and 26 times lower than that for HPr, respectively. Our results suggested that (i) the inefficiency of the phosphorylation of HPr(His∼P) by HprK/P results from the presence of a negative charge at position 15 as well as from other structural elements and (ii) the contribution of streptococcal HprK/P to the synthesis of HPr-P2 in vivo is marginal. [ABSTRACT FROM AUTHOR]

ABSTRACT: We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr• EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr•] intermediate has an electropositive and pH-dependent microenvironment [gx value of 2.0077(0) at pH ⩾ 8.0 and 2.0066(2) at 4.0 ⩽ pH ⩽ 7.5] possibly related to the radical stability and function. Two types of organic molecules (small aromatic vs bulkier substrates) allowed us to distinguish different mechanisms for substrate oxidation. [Fe(IV)=O Por•+] is the oxidizing species of benzohydroxamic acid, o-dianisidine, and o-anisidine via a heme-edge reaction and of mitoxantrone via a long-range electron transfer (favored at pH 8) not involving the tyrosyl radical, the formation of which competed with the substrate oxidation at pH 5. In contrast, the very efficient reaction with ABTS at pH 5 is consistent with [Fe(IV)0 Tyr] being the oxidizing species. Accordingly, the identification of the ABTS binding site by X-ray crystallography may be a valuable tool in rational drug design. [ABSTRACT FROM AUTHOR]

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 °C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (Tms) in concentrated rather than in dilute solutions (ΔTm ∼ 2–10 °C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower Tm than in dilute solutions (ΔTm ∼ 2–20 °C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions. [ABSTRACT FROM AUTHOR]

The transient kinetics of glucose binding to glucokinase (OK) was studied using stopped-flow fluorescence spectrophotometry to investigate the underlying mechanism of positive cooperativity of monomeric OK with glucose. Glucose binding to GK was shown to display biphasic kinetics that fit best to a reversible two-step mechanism. GK initially binds glucose to form a transient intermediate, namely, E*·glucose, followed by a conformational change to a catalytically competent Eglucose complex. The microscopic rate constants for each step were determined as follows: on rate k1 of 557 M-1 s-1 and off rate k-1 of 8.1 s-1 for E*·glucose formation, and forward rate k2 of 0.45 s-1 and reverse rate k-2 of 0.28 s-1 for the conformational change from E*·glucose to E·glucose. These results suggest that the enzyme conformational change induced by glucose binding is a reversible, slow event that occurs outside the catalytic cycle (kcat = 38 s-1). This slow transition between the two enzyme conformations modulated by glucose likely forms the kinetic foundation for the allosteric regulation. Furthermore, the kinetics of the enzyme conformational change was altered in favor of E·glucose formation in D2O, accompanied by a decrease in cooperativity with glucose (Hill slope of 1.3 in D2O vs 1.7 in H2O). The deuterium solvent isotope effects confirm the role of the conformational change in the magnitude of glucose cooperativity. Similar studies were conducted with GK activating mutation Y214C at the allosteric activator site that is likely involved in the protein domain rearrangement associated with glucose binding. The mutation enhanced equilibrium glucose binding by a combination of effects on both the formation of E*·glucose and an enzyme conformational change to E·glucose. Kinetic simulation by KINSIM supports the conclusion that the kinetic cooperativity of GK arises from slow glucose-induced conformational changes in GK. [ABSTRACT FROM AUTHOR]

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp 123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F1W123F human MnSOD at 1.95 Å resolution suggests that this effect is not related to a conformational change in the side chain of Gin 143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles. [ABSTRACT FROM AUTHOR]

β-Benzoyl-DL-alanine was synthesized from α-bromoacetophenone and diethyl acetamidomalonate. The racemic amino acid was resolved by carboxypeptidase A-catalyzed hydrolysis of the N-trifluoroacetyl derivative. β-Benzoyl-L-alanine is a good substrate of kynureninase from Pseudomonas fluorescens, with kcat and kcat/Km values of 0.7 s-1 and 8.0 × 104M-1, respectively, compared to kcat = 16.0 s-1 and kcat/Km = 6.0 × 105 M-1 s-1 for L-kynurenine. In contrast to the reaction of L-kynurenine, β-benzoyl-L-alanine does not exhibit a significant solvent isotope effect on kcat (Hk/Dk = 0.96 ± 0.06). The pre-steady-state kinetics of the reaction of β-benzoyl-L-alanine were investigated by rapid scanning stopped-flow spectrophotometry. The spectra show the formation of a quinonoid intermediate, with λmax = 490 nm, in the dead time of the instrument, which then decays, with k = 210 s-1, to form a transient intermediate with λmax at 348 nm. In the presence of benzaldehyde, the 348 nm intermediate decays, with k = 0.7 s-1, to form a quasistable quinonoid species with λmax = 492 nm. Previous studies demonstrated that benzaldehyde can trap an enamine intermediate formed after the Cβ-Cγ bond cleavage [Phillips, R. S., Sundararaju, B., and Koushik, S. V. (1998) Biochemistry 37, 8783-8789]. Thus, the 348 nm intermediate is kinetically competent. The position of the absorption maximum and shape of the band is consistent with a PMP-ketimine intermediate. The results from chemical quenching analysis do not show a burst of benzoate and, thus, also support the formation of benzoate as the rate-determining step. [ABSTRACT FROM AUTHOR]

Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s[sup-1]) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s[sup-1]) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s[sup-1]) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-[sup2]H]- NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP[sup+]. [ABSTRACT FROM AUTHOR]

The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, L-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate. [ABSTRACT FROM AUTHOR]

Analyzes the minimal structural requirements for Pin1 substrate recognition. Application of the panel of peptides; Use of direct UV-visible spectrophotometric detection of prolyl isomerization; Observation of weak competitive inhibition of Pin1.

Examines the role of electrostatics in cytochrome f and cyanobacterium phormidium laminosum plastocyanin interaction. Effects of mutation on midpoint potentials; Use of stopped-flow spectrophotometry in determining overall rate constant of reaction dependence; Consideration of electrostatics as the major contributor to k[sub 2] in plants.

Presents a study which used fluorescence spectrophotometry and stopped-flow absorbance to investigate how the oxidation of urate is catalyzed by soybean urate oxidase. Information on urate oxidase; Details on the urate oxidase reaction in tropical leguminous plants; Method used for the study; What was observed in the study.

Presents a study which focuses on the stability of the manganese (Mn)II binding to manganese peroxidase (MnP) with relation to the function of pH by the spectrophotometric and potentiometric titrations. What the sensitivity of the potentiometric titrations allows for; Information on the MnP.

Presents a study on the reaction of CO and O2 with reduced cytochrome caa3 from bacillus subtilis, using the rapid reaction spectrophotometry. Reaction of a reduced caa3 with CO; Effect of kinetic constants on the CO complex of 0.83 muM; Kinetic simulation of the single turnover of reduced cytochrome caa3 with O2.

Determines the on-rate and off-rate for the binding of carbon monoxide (CO) to the heme of soluble guanylate cyclase (sGC) using stopped-flow spectrophotometry. Comparison of the equilibrium dissociation constant with that calculated from the rate constants; Indication that the binding of CO to sGC is a simple one-step process.

Presents a study developing a method to determine the acylation and deacylation elementary rate constants using electrospray mass spectrometry and ultraviolet spectrophometry. Results of hydrolysis of penicillin; Cause of loss of activity against penicillins in the G238S mutant; Modifications of elementary rate constants.

Reports on the kinetic studies of spinach carbonic anhydrase (CA) using stopped-flow spectrophotometry at steady state and 13C-NMR exchange at equilibrium. Measurement of pH-independent solvent deuterium isotope; Comparison with CA structures in plants and animals; Experimental procedures and results.

Investigates the proton slip of the chloroplast adenosine triphosphatase (ATPase). Study of charge flow through ATPase by flash spectrophotometry; Proton slip inhibition by nucleotide, phosphate, arsenate and insufficient proton motive force; Entropic machine model.