Purple Intermediate Research Articles

A bis-Phenolate Carbene-Supported bis-μ-Oxo Iron(IV/IV) Complex with a [FeIV(μ-O)2FeIV] Diamond Core Derived from Dioxygen Activation.

The diiron(II) complex, [(OCO)Fe(MeCN)]2 (1, MeCN = acetonitrile), supported by the bis-phenolate carbene pincer ligand, 1,3-bis(3,5-di-tert-butyl-2-hydroxyphenyl)benzimidazolin-2-ylidene (OCO), was synthesized and characterized by single-crystal X-ray diffraction, 1H nuclear magnetic resonance, infrared (IR) vibrational, ultraviolet/visible/near-infrared (UV/vis/NIR) electronic absorption, 57Fe Mössbauer, X-band electron paramagnetic resonance (EPR) and SQUID magnetization measurements. Complex 1 activates dioxygen to yield the diferric, μ-oxo-bridged complex [(OCO)Fe(py)(μ-O)Fe(O(C═O)O)(py)] (2) that was isolated and fully characterized. In 2, one of the iron-carbene bonds was oxidized to give a urea motif, resulting in an O(CNHC═O)O binding site, while the other Fe(OCO) unit remained unchanged. When the reaction is performed at -80 °C, an intensively colored, purple intermediate is observed (INT, λmax = 570 nm; ε = 5600 mol L-1 cm-1). INT acts as a sluggish oxidant, reacting only with easily oxidizable substrates, such as PPh3 or 2-phenylpropionic aldehyde (2-PPA). The identity of INT can be best described as a dinuclear complex containing a closed diamond core motif [(OCO)FeIV(μ-O)2FeIV(OCO)]. This proposal is based on extensive spectroscopic [UV/vis/NIR electronic absorption, 57Fe Mössbauer, X-band EPR, resonance Raman (rRaman), X-ray absorption, and nuclear resonance vibrational (NRVS)] and computational studies. The conversion of the diiron(II) complex 1 to the oxo diiron(IV) intermediate INT is reminiscent of the O2 activation process in soluble methane monooxygenases (sMMO). Most importantly, the low reactivity of INT supports the consensus that the [FeIV(μ-O)2FeIV] diamond core in sMMO is kinetically inert and needs to open up to terminal FeIV═O cores to react with the strong C-H bonds of methane.

Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry.

Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxidative half-reactions using dimethylglycine and O2 as substrates. The reductive half-reaction is triphasic. The rate of the fast phase is dependent on substrate concentration, involves flavin reduction, and has a limiting rate constant of 244 s(-1). This phase also displays a kinetic isotope effect of 2.9. Completion of the first kinetic phase generates an intermediate with broad spectral signature between 350 and 500 nm, which is attributed to a reduced enzyme-iminium charge-transfer species, similar to the purple intermediate that accumulates in reactions of D-amino acid oxidase (DAAO) with alanine. The second phase (16 s(-1)) is independent of substrate concentration and is attributed to iminium hydrolysis/deprotonation. The third phase (2 s(-1)) is attributed to product release, the rate of which is less than the steady-state turnover rate (10.6 s(-1)). Flavin oxidation of dithionite- and dimethylglycine-reduced enzyme by O2 occurs in a single phase, and the rate shows a linear dependence on oxygen concentration, giving bimolecular rate constants of 342 and 201 mM(-1) x s(-1), respectively. Enzyme-monitored turnover experiments indicate that decay of the reduced enzyme-iminium intermediate is rate-limiting, consistent with rate constants determined from single turnover studies. A minimal kinetic mechanism is presented, which establishes a close relationship to the mechanism of action of DAAO. The covalent flavin in dimethylglycine oxidase is identified as an alphaN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics.

Three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase. Optimization of the oxidative half-reaction through alignment of the product with reduced flavin.

The three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, delta(1)-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The (+)HN=C double bond of DPC overlaps the N(5)-C(4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C(4a), thereby augmenting the electron density at C(4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.

Resonance Raman study on reduced flavin in purple intermediate of flavoenzyme: use of [4-carbonyl-18O]-enriched flavin.

4-Carbonyl-18O]-enriched lumiflavin, riboflavin, and FMN were prepared by incubating each corresponding non-labeled flavin in 1 M Na18OH (H218O) at 25 degrees C. [4-Carbonyl-18O]FAD was prepared from the corresponding riboflavin by using FAD synthetase. Isotope effects by [4-carbonyl-18O]-labeling confirmed that the 1,709-cm-1 band in the IR spectrum of lumiflavin and the 1,711-cm-1 band in the Raman spectrum of FAD are mainly derived from C(4)=O stretching vibrational mode. The 1,605-cm-1 Raman band of the anionic reduced flavin in the purple intermediate of D-amino acid oxidase (DAO) with D-proline or D-alanine does not shift in DAO reconstituted with [4-carbonyl-18O]FAD, although it shifts with [4,10a-13C2]- or [4a-13C]FAD. Thus the band is mainly due to the C(4a)=C(10a) stretching vibrational mode and includes no contribution from C(4)=O stretching vibration. The band frequencies cover a fairly wide range (1,602-1,620 cm-1) depending on the enzymes. The frequencies of the reduced flavin in the purple intermediates of the dehydrogenases (medium-chain acyl-CoA, short-chain acyl-CoA, and isovaleryl-CoA dehydrogenases) are higher than those of the oxidases (DAO and L-phenylalanine oxidase). This indicates that the C(4a)=C(10a) bond order of reduced flavin in the dehydrogenases with the low reactivity for molecular oxygen is stronger than that in the oxidases with high reactivity. Therefore, the band frequency of C(4a)=C(10a) stretching may serve as an indicator of the reactivity of flavoprotein with molecular oxygen. Furthermore, strong hydrogen bonding of flavin at the N(1) moiety with the hydroxyl group of Thr136 in MCAD is probably responsible for the strong bond of the C(4a)=C(10a) of reduced flavin in the dehydrogenase.

Functional models for iron-bleomycin

A new pentadentate ligand, N,N-bis(2-pyridylmethyl)- N-bis(2-pyridyl)methylamine (N4Py), has been prepared. The corresponding iron(II) complex, [N4PyFe(CH 3CN)](ClO 4) 2, serves as a functional model for the active site of iron-bleomycin. Upon treatment with H 2O 2 the formation of a purple intermediate has been observed, which has been characterized as an iron(III)-hydroperoxide complex. This N4Py-iron-hydroperoxide system has proven to be an active oxidation catalyst.

Studies on Phe-228 and Leu-307 Recombinant Mutants of Porcine Kidney D-Amino Acid Oxidase: Expression, Purification, and Characterization1

Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Complex formation between reduced D-amino acid oxidase and pyridine carboxylates.

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.

COMPARATIVE REACTIVITIES OF SELENOSULFATES AND SELENENYLTHIOSULFATES WITH THIOLS

Abstract Nitroaryl selenosulfates (1) and selenenylthiosulfates (2) reacted instantaneously with a thiol. N-succinylcysteamine. in water at pH 4.6 to give an insoluble selenenyl sulfide and diselenide. The selenenyl sulfide was the major product from the 2-nitro compounds whereas the diselenide was the major (almost exclusive) product from the 4-nitro compounds. However when the 4-nitro compounds reacted with N-succinylcysteamine in dimethylformamide the selenenyl sulfide was the major and the diselenide the minor product indicating the formation of selenenyl sulfide preceded that of diselenide. Also in the dimethylformamide reactions a short-lived, light-sensitive, purple intermediate was formed which was identified as 4-nitroselenophenol. It was impossible to compare reactivities of selenosulfates and selenenylthiosulfates in water since all reacted immediately to form insoluble products. However, when formation of the purple complex of 4-nitroselenophenol in dimethylformamide was used as an indicator, ...

Resonance Raman study on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm −1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm −1 in the RR spectrum with D-alanine was shifted to 1675 cm −1 upon [ 15N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm −1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.

Lifetime of the purple intermediate of D-amino acid oxidase under anaerobic conditions.

The lifetime of the purple intermediate formed from D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] and a neutral D-amino acid under anaerobic conditions was measured with a series of neutral D-amino acids. The lifetime increases with increase in the number of carbons in the side chain of the amino acid up to 4-5 atoms and then decreases with further increase in the number of carbons. This suggests that the hydrophobicity of the alkyl group of the neutral D-amino acid determines the lifetime of the purple intermediate unless a steric effect occurs.

Effect of alcohols on the structure and function of D-amino-acid oxidase.

The absorption spectrum of D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) was significantly perturbed by various alcohols; typical fine structures were observed in the visible absorption bands, accompanied by blue shifts of the peaks. Both fluorescence intensity and fluorescence polarization were increased upon the addition of alcohols, indicating that the coenzyme is not liberated from the apoenzyme but the hydrophobicity of the environment of the enzyme-bound flavin is increased. Upon the addition of alcohols, the circular dichroism of the enzyme was markedly modified in the visible and near-ultraviolet regions, while that of the apoenzyme in the near- and far-ultraviolet regions was scarcely modified, indicating a change in the interaction between the flavin coenzyme and protein. Both the apparent maximal velocity and the apparent Michaelis constant of the enzyme were increased by the addition of alcohols. The presence of alcohols tends to dissociate the dimer of this enzyme into the monomer, but the dissociation does not fully explain the increase in the maximal velocity of the enzyme by alcohols, because the increase in the maximal velocity caused by alcohols is larger than that expected from the dissociation. Since the rate of formation of the purple intermediate was decreased by alcohols in both the dimer and the monomer, the increase in the maximal velocity could be ascribed to an increase in the rate of dissociation of the enzyme-product complex. This increase could be ascribed to the protein conformational change, which is probably provoked by combination of alcohols with the enzyme at a locus other than that for substrate binding.

Mechanism of enzyme action. VI. Kinetic isotope effect on d-amino acid oxidase reaction

1. 1. The rate of formation of the purple itermediate of d-amino acid oxidase [ d-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3] in the anaerobic reaction of this enzyme with its substrate was reduced by substitution of the α-hydrogen of the substrate for deuterium. This indicates that removal of the α-hydrogen of the substrate occurs prior to or in concert with the formation of the purple intermediate. The extent of the deuterium kinetic isotope effect differed for different substrates. 2. 2. The α-deuteration of the substrate affected neither the rate of conversion of the purple intermediate to the fully reduced enzyme nor the rate of oxidation of the purple intermediate with O 2. 3. 3. The kinetic isotope effect on the catalytic oxidation of the substrate was less than that on the formation of the purple intermediate. This indicates that the purple intermediate formation partially determines the overall rate of the enzymic reaction, but the effect on the overall rate is thought to result merely from the effect on the process of the purple intermediate formation. The kinetic isotope effect on the catalytic oxidation was increased by decreasing the enzyme concentration. This may be interpreted to mean that the process of the purple intermediate formation is more rate controlling in the monomeric enzyme than in the dimeric enzyme.

Structure and function of D-amino acid oxidase. VI. Relation between the quaternary structure and the catalytic activity.

1. The maximum velocity (Vmax/e0) of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] obtained by increasing the substrate concentration in the presence of 2 M urea was found to be higher than that obtained in the absence of urea, though urea inhibited the enzymic reaction in competition with the substrate. The acceleration of the catalytic activity was ascribed to the dissociation of the dimer of the enzyme into its monomer. 2. Upon lowering the concentration of the enzyme, the value of Km was found to be increased, besides the acceleration of Vmax//e0 value, indicating that the Km value is larger in the monomer than in the dimer. 3. The purple complex prepared in the presence of 2 M urea gave the same absorption spectrum with that prepared in the absence of urea. However, they gave different values of s020, w; 3.5 S for the former and 6.9 S for the latter, indicating the occurrence of the purple intermediate of monomeric form during the catalytic reaction of the enzyme with neutral amino acids such as D-alanine in the presence of 2 M urea. 4. The acceleration of the catalytic activity in the monomer was considered to occur after the formation of the purple intermediate, most probably during the liberation of the product, since the rate of formation of the purple intermediate was larger in the dimer than in the monomer.

A characterization of a purple intermediate of D-amino acid oxidase.

Journal Article A Characterization of a Purple Intermediate of D-Amino Acid Oxidase Get access KUNIO YAGI, KUNIO YAGI Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar KENTARO OKAMURA, KENTARO OKAMURA Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar MAKOTO NAOI, MAKOTO NAOI Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar AKIRA KOTAKI AKIRA KOTAKI Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 59, Issue 5, May 1966, Pages 527–530, https://doi.org/10.1093/oxfordjournals.jbchem.a128338 Published: 01 May 1966 Article history Received: 19 February 1966 Published: 01 May 1966