Quinone reductase reaction catalyzed by streptococcus faecalis NADH peroxidase

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Abstract

NADH peroxidase is a flavoenzyme having a single redox-active thiol, Cys42, that cycles between sulfenate and thiol forms in the NADH-dependent reduction of hydrogen peroxide. NADH peroxidase catalyzes the NADH-dependent reduction of quinones with turnover numbers between 1.2 and 3.9 s-1, per mole of FAD, at pH 7.5. The bimolecular rate constants for quinone reduction, V/K, ranged from 4.3 × 103 to 6.0 × 105 M-1 s-1 for 14 quinones whose redox potentials varied between -0.41 and 0.09 V. The logarithms of the V/K values for these quinones are hyperbolically dependent on their single-electron reduction potentials (E71). One-electron reduction of benzoquinone accounts for about 50% of the total electron transfer catalyzed by NADH peroxidase at pH 7, with the remainder of the reduction being catalyzed by a two-electron (hydride) transfer. Cys42 can be irreversibly oxidized to the sulfonate by hydrogen peroxide, with inactivation of the peroxidatic activity of the enzyme. The residual quinone reductase activity of NADH peroxidase which has undergone oxidative inactivation of the active site Cys42 indicates that this residue is not involved in the reduction of the quinones. Product inhibition studies suggest the possibility of overlap of the pyridine nucleotide and quinone binding sites in the reduced enzyme at low pH values. The pH dependence of the maximum velocity of naphthoquinone reduction shows that deprotonation of an enzymic group, exhibiting a pK value of ca. 6.2, decreases the maximal velocity. Primary deuterium kinetic isotope effects on V and V/K for quinone-dependent NADH oxidation increase upon protonation of a group, exhibiting a pK value of 6.4. These data are most consistent with a change in the rate-limiting step and the mechanism of quinone reduction as the pH is lowered. We suggest that these data can be accommodated in a model in which the ionization of the tightly-bound FAD affects the enzymes' affinity for nucleotides, and in which two-electron reduction of quinones occurs in the non-nucleotide-liganded enzyme. The data suggest that at neutral pH, where NADH binding is tight, quinones must occupy a distinct site and be reduced by long-range electron transfer.

Original languageEnglish (US)
Pages (from-to)6621-6627
Number of pages7
JournalBiochemistry®
Volume34
Issue number20
StatePublished - 1995

Fingerprint

NAD+ peroxidase
NAD(P)H Dehydrogenase (Quinone)
Quinones
Enterococcus faecalis
Electrons
NAD
Flavin-Adenine Dinucleotide
Enzymes
Sulfhydryl Compounds
Hydrogen Peroxide
Oxidation-Reduction
Nucleotides
Naphthoquinones
Deuterium
Isotopes
Catalytic Domain
Binding Sites
Deprotonation
benzoquinone
Protonation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quinone reductase reaction catalyzed by streptococcus faecalis NADH peroxidase. / Blanchard, John S.

In: Biochemistry®, Vol. 34, No. 20, 1995, p. 6621-6627.

Research output: Contribution to journalArticle

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abstract = "NADH peroxidase is a flavoenzyme having a single redox-active thiol, Cys42, that cycles between sulfenate and thiol forms in the NADH-dependent reduction of hydrogen peroxide. NADH peroxidase catalyzes the NADH-dependent reduction of quinones with turnover numbers between 1.2 and 3.9 s-1, per mole of FAD, at pH 7.5. The bimolecular rate constants for quinone reduction, V/K, ranged from 4.3 × 103 to 6.0 × 105 M-1 s-1 for 14 quinones whose redox potentials varied between -0.41 and 0.09 V. The logarithms of the V/K values for these quinones are hyperbolically dependent on their single-electron reduction potentials (E71). One-electron reduction of benzoquinone accounts for about 50{\%} of the total electron transfer catalyzed by NADH peroxidase at pH 7, with the remainder of the reduction being catalyzed by a two-electron (hydride) transfer. Cys42 can be irreversibly oxidized to the sulfonate by hydrogen peroxide, with inactivation of the peroxidatic activity of the enzyme. The residual quinone reductase activity of NADH peroxidase which has undergone oxidative inactivation of the active site Cys42 indicates that this residue is not involved in the reduction of the quinones. Product inhibition studies suggest the possibility of overlap of the pyridine nucleotide and quinone binding sites in the reduced enzyme at low pH values. The pH dependence of the maximum velocity of naphthoquinone reduction shows that deprotonation of an enzymic group, exhibiting a pK value of ca. 6.2, decreases the maximal velocity. Primary deuterium kinetic isotope effects on V and V/K for quinone-dependent NADH oxidation increase upon protonation of a group, exhibiting a pK value of 6.4. These data are most consistent with a change in the rate-limiting step and the mechanism of quinone reduction as the pH is lowered. We suggest that these data can be accommodated in a model in which the ionization of the tightly-bound FAD affects the enzymes' affinity for nucleotides, and in which two-electron reduction of quinones occurs in the non-nucleotide-liganded enzyme. The data suggest that at neutral pH, where NADH binding is tight, quinones must occupy a distinct site and be reduced by long-range electron transfer.",
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N2 - NADH peroxidase is a flavoenzyme having a single redox-active thiol, Cys42, that cycles between sulfenate and thiol forms in the NADH-dependent reduction of hydrogen peroxide. NADH peroxidase catalyzes the NADH-dependent reduction of quinones with turnover numbers between 1.2 and 3.9 s-1, per mole of FAD, at pH 7.5. The bimolecular rate constants for quinone reduction, V/K, ranged from 4.3 × 103 to 6.0 × 105 M-1 s-1 for 14 quinones whose redox potentials varied between -0.41 and 0.09 V. The logarithms of the V/K values for these quinones are hyperbolically dependent on their single-electron reduction potentials (E71). One-electron reduction of benzoquinone accounts for about 50% of the total electron transfer catalyzed by NADH peroxidase at pH 7, with the remainder of the reduction being catalyzed by a two-electron (hydride) transfer. Cys42 can be irreversibly oxidized to the sulfonate by hydrogen peroxide, with inactivation of the peroxidatic activity of the enzyme. The residual quinone reductase activity of NADH peroxidase which has undergone oxidative inactivation of the active site Cys42 indicates that this residue is not involved in the reduction of the quinones. Product inhibition studies suggest the possibility of overlap of the pyridine nucleotide and quinone binding sites in the reduced enzyme at low pH values. The pH dependence of the maximum velocity of naphthoquinone reduction shows that deprotonation of an enzymic group, exhibiting a pK value of ca. 6.2, decreases the maximal velocity. Primary deuterium kinetic isotope effects on V and V/K for quinone-dependent NADH oxidation increase upon protonation of a group, exhibiting a pK value of 6.4. These data are most consistent with a change in the rate-limiting step and the mechanism of quinone reduction as the pH is lowered. We suggest that these data can be accommodated in a model in which the ionization of the tightly-bound FAD affects the enzymes' affinity for nucleotides, and in which two-electron reduction of quinones occurs in the non-nucleotide-liganded enzyme. The data suggest that at neutral pH, where NADH binding is tight, quinones must occupy a distinct site and be reduced by long-range electron transfer.

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