Substrate-mediated oxygen activation by homoprotocatechuate 2,3-dioxygenase

Intermediates formed by a tyrosine 257 variant

Michael M. Mbughuni, Katlyn K. Meier, Eckard Münck, John D Lipscomb

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O2 bind in adjacent ligand sites of the active site FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the FeII to O2 gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases kcat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F Int1 HPCA and Y257FInt2 HPCA, accumulate during the Y257F-HPCA + O2 reaction prior to formation of the ring-cleaved product. Y257FInt1 HPCA is colorless and is formed as O2 binds reversibly to the HPCA-enzyme complex. Y257FInt2 HPCA forms spontaneously from Y257F Int1 HPCA and displays a chromophore at 425 nm (ε425 = 10 500 M-1 cm-1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain FeII. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of FeII, and the low O2 affinity suggest that Y257FInt1 HPCA is an HPCA-FeII-O2 complex with little electron delocalization onto the O2. In contrast, the intense spectrum of Y257FInt2 HPCA suggests the intermediate is most likely an HPCA quinone-FeII-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O2.

Original languageEnglish (US)
Pages (from-to)8743-8754
Number of pages12
JournalBiochemistry
Volume51
Issue number44
DOIs
StatePublished - Nov 6 2012

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3,4-dihydroxyphenylacetate 2,3-dioxygenase
Tyrosine
Chemical activation
Electrons
Chromophores
Oxygen
Catalytic Domain
Hydrogen bonds
Substrates
Reaction intermediates
Hydrogen Bonding
Coulomb interactions
Static Electricity
Hydroxyl Radical
Hydrogen
Crystal structure
Ligands
Amino Acids
Mutation
Kinetics

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Substrate-mediated oxygen activation by homoprotocatechuate 2,3-dioxygenase : Intermediates formed by a tyrosine 257 variant. / Mbughuni, Michael M.; Meier, Katlyn K.; Münck, Eckard; Lipscomb, John D.

In: Biochemistry, Vol. 51, No. 44, 06.11.2012, p. 8743-8754.

Research output: Contribution to journalArticle

Mbughuni, Michael M. ; Meier, Katlyn K. ; Münck, Eckard ; Lipscomb, John D. / Substrate-mediated oxygen activation by homoprotocatechuate 2,3-dioxygenase : Intermediates formed by a tyrosine 257 variant. In: Biochemistry. 2012 ; Vol. 51, No. 44. pp. 8743-8754.
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abstract = "Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O2 bind in adjacent ligand sites of the active site FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the FeII to O2 gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases kcat by about 75{\%}. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F Int1 HPCA and Y257FInt2 HPCA, accumulate during the Y257F-HPCA + O2 reaction prior to formation of the ring-cleaved product. Y257FInt1 HPCA is colorless and is formed as O2 binds reversibly to the HPCA-enzyme complex. Y257FInt2 HPCA forms spontaneously from Y257F Int1 HPCA and displays a chromophore at 425 nm (ε425 = 10 500 M-1 cm-1). M{\"o}ssbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain FeII. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of FeII, and the low O2 affinity suggest that Y257FInt1 HPCA is an HPCA-FeII-O2 complex with little electron delocalization onto the O2. In contrast, the intense spectrum of Y257FInt2 HPCA suggests the intermediate is most likely an HPCA quinone-FeII-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O2.",
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AU - Münck, Eckard

AU - Lipscomb, John D

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N2 - Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O2 bind in adjacent ligand sites of the active site FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the FeII to O2 gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases kcat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F Int1 HPCA and Y257FInt2 HPCA, accumulate during the Y257F-HPCA + O2 reaction prior to formation of the ring-cleaved product. Y257FInt1 HPCA is colorless and is formed as O2 binds reversibly to the HPCA-enzyme complex. Y257FInt2 HPCA forms spontaneously from Y257F Int1 HPCA and displays a chromophore at 425 nm (ε425 = 10 500 M-1 cm-1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain FeII. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of FeII, and the low O2 affinity suggest that Y257FInt1 HPCA is an HPCA-FeII-O2 complex with little electron delocalization onto the O2. In contrast, the intense spectrum of Y257FInt2 HPCA suggests the intermediate is most likely an HPCA quinone-FeII-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O2.

AB - Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O2 bind in adjacent ligand sites of the active site FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the FeII to O2 gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases kcat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F Int1 HPCA and Y257FInt2 HPCA, accumulate during the Y257F-HPCA + O2 reaction prior to formation of the ring-cleaved product. Y257FInt1 HPCA is colorless and is formed as O2 binds reversibly to the HPCA-enzyme complex. Y257FInt2 HPCA forms spontaneously from Y257F Int1 HPCA and displays a chromophore at 425 nm (ε425 = 10 500 M-1 cm-1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain FeII. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of FeII, and the low O2 affinity suggest that Y257FInt1 HPCA is an HPCA-FeII-O2 complex with little electron delocalization onto the O2. In contrast, the intense spectrum of Y257FInt2 HPCA suggests the intermediate is most likely an HPCA quinone-FeII-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O2.

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