Use of Free Energy Relationships To Probe the Individual Steps of Hydroxylation of <i>p</i>-Hydroxybenzoate Hydroxylase:  Studies with a Series of 8-Substituted Flavins

Ortiz-Maldonado, Mariliz; Ballou, David P.; Massey, Vincent

doi:10.1021/bi990560e

Cited by 52 publications

(92 citation statements)

References 54 publications

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“…The redox potential of the enzyme-bound flavin is uniformly more positive than that of the free flavin, indicating a higher affinity of the reduced flavin to the enzyme than that of the oxidized form. The slope of Ϸ1.3 is almost identical to that found with many of the same flavins bound to p-hydroxybenzoate hydroxylase (21) and shows that the more electron-withdrawing the substituent is, the stronger the preference for the enzyme to bind the reduced flavin.…”

Section: Correlation Of the Redox Midpoint Potentials Between Free Andsupporting

confidence: 67%

“…The slope of the plot where the observed rate constant depends on redox potential yields a Hammett value of ϷϪ2.5, consistent with a decrease of negative charge in conversion of the anionic reduced flavin to the neutral oxidized flavin, and again consistent with a late transition state. The reactivity profile with oxygen is similar to that found with phydroxybenzoate hydroxylase, including the breakpoint at approximately the same flavin redox potential (21).…”

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confidence: 66%

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On the interpretation of quantitative structure–function activity relationship data for lactate oxidase

Yorita

Misaki

Palfey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The native flavin, FMN, has been removed from the L-lactate oxidase of Aerococcus viridans, and the apoprotein reconstituted with 12 FMN derivatives with various substituents at the flavin 6-and 8-positions. Impressive linear relationships are exhibited between the sum of the Hammett para and ortho parameters and the redox potentials of the free flavins, and between the redox potentials of the free and enzyme-bound flavins. Rapid reaction kinetics studies of the reconstituted enzymes with the substrates L-lactate and L-mandelate show an increase in the reduction rate constant with increasing redox potential, except that, with lactate, a limiting rate constant of Ϸ700 s ؊1 is obtained with flavins of high potential. Similar breakpoints are found in plots of the rate constants for flavin N5-sulfite adduct formation and for the reaction of the reduced enzymes with molecular oxygen. These results are interpreted in terms of a two-step equilibrium preceding the chemical reaction step, in which the second equilibrium step provides an upper limit to the rate with which the particular substrate or ligand is positioned with the flavin in the correct fashion for the observed chemical reaction to occur. The relationship of rate constants for flavin reduction and N5-sulfite adduct formation with flavin redox potential below the observed breakpoint indicate development of significant negative charge in the transition states of the reactions. In the case of reduction by substrate, the results are consistent either with a hydride transfer mechanism or with the so called ''carbanion'' mechanism, in which the substrate ␣-proton is abstracted by an enzyme base protected from exchange with solvent. These conclusions are supported by substrate ␣-deuterium isotope effects and by solvent viscosity effects on sulfite binding. L -lactate oxidase (LOX) from Aerococcus viridans is a member of the family of FMN-containing enzymes that catalyze the oxidation of ␣-hydroxyacids (1). Considerable evidence has been accumulated that the dehydrogenation reaction involved proceeds via a carbanion mechanism (see refs. 2 and 3 for reviews). The recent crystal structure determination of D-amino acid oxidase (4, 5), a related enzyme involved in the oxidation of ␣-amino acids, has led to a reconsideration of its reaction mechanism, which, like that of the ␣-hydroxyacid oxidases, had been considered to involve a carbanion mechanism. Within the ␣-hydroxyacid oxidase family, glycolate oxidase and flavocytochrome b 2 are the only enzymes whose crystal structures have been solved (6, 7). Unlike D-amino acid oxidase, which lacks an active site base capable of abstraction of the ␣-proton from the substrate, and with which a hydride transfer mechanism now appears likely (4, 8), both glycolate oxidase and flavocytochrome b 2 have a histidine residue that appears to be positioned suitably to function as an active site base. This histidine and a set of other residues are conserved in all members of the ␣-hydroxyacid oxidase family. We have reported a preliminary...

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Section: Correlation Of the Redox Midpoint Potentials Between Free Andsupporting

confidence: 67%

supporting

confidence: 66%

On the interpretation of quantitative structure–function activity relationship data for lactate oxidase

Yorita

Misaki

Palfey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…An exception to this was found in the reaction of phenol hydroxylase (27) in which the enzyme showed formation of C4a-peroxyflavin anion before protonation to C4a-hydroperoxyflavin. The latter species is required in these aromatic hydroxylases because their reactions are involved in electrophilic aromatic substitution in which the C4a-hydroperoxyflavin acts as an electrophile (4,7,9). Although the advantage of having discrete steps of C4a-peroxyflavin and C4a-hydroperoxyflavin formation in 3HB6H and phenol hydroxylase is not clear, it highlights the differences in the proton transfer pathways among flavin-dependent hydroxylases.…”

Section: Discussionmentioning

confidence: 99%

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

Sucharitakul

Tongsook

Pakotiprapha

et al. 2013

Journal of Biological Chemistry

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Background: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a flavoprotein hydroxylase catalyzing the para-hydroxylation of 3-hydroxybenzoate. Results: The oxidative half-reaction was studied using transient kinetics. The enzyme reaction mechanism was elucidated. Conclusion:The product release and the hydroxylation limit the turnovers and the enzyme shows characteristics different from the ortho-hydroxylation enzymes. Significance: This is the first report on the oxygenation mechanism of a para-hydroxylating flavoenzyme.

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“…The former case is observed in Baeyer-Villiger monooxygenases (such as cyclohexanone monooxygenase) (19) or bacterial luciferase (20) in which the flavin C4a-peroxide acts as a nucleophile attacking the substrate. On the contrary, a flavin C4a-hydroperoxide has been well documented in flavin-containing monooxygenases (21) and aromatic hydroxylases (22)(23)(24), in which the terminal oxygen of hydroperoxyflavin acts as an electrophile in an aromatic substitution reaction. The three-dimensional structure suggests that an electrophilic aromatic substitution mechanism is likely to occur also in the C 2 reaction.…”

Section: Discussionmentioning

confidence: 99%

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

Alfieri

Fersini

Ruangchan

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

133

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p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADHdependent FMN reductase and a monooxygenase (C 2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C 2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C 2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C 2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-Å radius and a 29-Å 3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. F lavoprotein monooxygenases use dioxygen to insert an oxygen atom into a substrate and have been found to be involved in a wide variety of biological reactions (1-3). The fundamental property of these enzymes is their ability to promote formation and stabilization of the C4a-hydroperoxyflavin (Fig. 1a) resulting from the reaction of the protein-bound reduced flavin with dioxygen. This key intermediate donates an oxygen atom to the substrate, generating the unstable C4a-hydroxyflavin that eliminates one molecule of water to yield oxidized flavin (5). Understanding the structural bases governing functional properties of monooxygenases is crucial to address one of the most fascinating issues in flavoenzymology: the ability of flavoenzymes to differentially react with molecular oxygen.In recent years, a new group of flavoprotein monooxygenases has been identified. These enzymes consist of two components: a reductase generating reduced flavin and a hydroxylase using reduced flavin to catalyze substrate monooxygenation (6). phydroxyphenylacetate hydroxylase from Acinetobacter baumannii catalyzes hydroxylation of p-hydroxyphenylacetate (HPA) to 3,4-dihydroxyphenylacetate (Fig. 1a). HPA hydroxylase has unusual features in both sequence and catalysis. The smaller reductase component of HPA hydroxylase (C 1 ) performs HPA-stimulated NADH-dependent reduction of free FMN, which is subsequently transferred to the larger monooxygenase component (C 2 ) and used for reaction with dioxygen and HPA monooxygenation (Fig. 1b). Specificity for FMN is conferred by C 1 , whereas C 2 works equally well with both reduced FMN (FMNH Ϫ ; Fig. 1a) and reduced FAD (7-10). C 2 can effectively stabilize the C4a-hydroperoxyflavin intermediate for minutes, and, at high concentration of HPA, a stable dead-end complex between C4a-hydroxyflavin and HPA is observed.Here, we present crystal structures of C 2 in the apoenzyme form and of its complexes with FMNH Ϫ (C 2 :FMNH Ϫ ) and HPA (C 2 :FMNH Ϫ :HPA). Structural analysis reported here provides a fram...

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Use of Free Energy Relationships To Probe the Individual Steps of Hydroxylation of p-Hydroxybenzoate Hydroxylase: Studies with a Series of 8-Substituted Flavins

Cited by 52 publications

References 54 publications

On the interpretation of quantitative structure–function activity relationship data for lactate oxidase

On the interpretation of quantitative structure–function activity relationship data for lactate oxidase

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

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