Limits...
Reduction of hydrophilic ubiquinones by the flavin in mitochondrial NADH:ubiquinone oxidoreductase (Complex I) and production of reactive oxygen species.

King MS, Sharpley MS, Hirst J - Biochemistry (2009)

Bottom Line: Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as rotenone and piericidin A).Here, we show that inhibitor-insensitive ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the flavin mononucleotide cofactor in the active site for NADH oxidation.The factors which determine the balance of reactivity between the two sites of ubiquinone reduction (the energy-transducing site and the flavin site) and the implications for mechanistic studies of ubiquinone reduction by complex I are discussed.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound enzyme that contains 45 different subunits, a non-covalently bound flavin mononucleotide, and eight iron-sulfur clusters. The mechanisms of NADH oxidation and intramolecular electron transfer by complex I are gradually being defined, but the mechanism linking ubiquinone reduction to proton translocation remains unknown. Studies of ubiquinone reduction by isolated complex I are problematic because the extremely hydrophobic natural substrate, ubiquinone-10, must be substituted with a relatively hydrophilic analogue (such as ubiquinone-1). Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as rotenone and piericidin A). Here, we show that inhibitor-insensitive ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the flavin mononucleotide cofactor in the active site for NADH oxidation. Moreover, semiquinones produced at the flavin site initiate redox cycling reactions with molecular oxygen, producing superoxide radicals and hydrogen peroxide. The ubiquinone reactant is regenerated, so the NADH:Q reaction becomes superstoichiometric. Idebenone, an artificial ubiquinone showing promise in the treatment of Friedreich's Ataxia, reacts at the flavin site. The factors which determine the balance of reactivity between the two sites of ubiquinone reduction (the energy-transducing site and the flavin site) and the implications for mechanistic studies of ubiquinone reduction by complex I are discussed. Finally, the possibility that the flavin site in complex I catalyzes redox cycling reactions with a wide range of compounds, some of which are important in pharmacology and toxicology, is discussed.

Show MeSH

Related in: MedlinePlus

Mechanism of Quinone Reduction at the Flavin Active Site, Incorporating both the Ping-Pong and Ping-Pong-Pong Reactions, and Double-Substrate InhibitionThe ping-pong and ping-pong-pong reactions diverge at [FlRed Q]: the ping-pong reaction follows the dotted arrow to produce QH2 and returns directly to [FlOx], and the ping-pong-pong reaction follows the outside pathway to form two semiquinones (QH•). Abbreviations: FlOx/Red, oxidized or reduced state of the flavin; terms in brackets, enzyme-bound species; kcat, first-order rate constant for substrate transformation and product dissociation; KM, Michaelis−Menten constant; KOx, KSemi, and KRed, dissociation constants, referring to the oxidized, semireduced, and reduced enzyme states, respectively.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651670&req=5

sch1: Mechanism of Quinone Reduction at the Flavin Active Site, Incorporating both the Ping-Pong and Ping-Pong-Pong Reactions, and Double-Substrate InhibitionThe ping-pong and ping-pong-pong reactions diverge at [FlRed Q]: the ping-pong reaction follows the dotted arrow to produce QH2 and returns directly to [FlOx], and the ping-pong-pong reaction follows the outside pathway to form two semiquinones (QH•). Abbreviations: FlOx/Red, oxidized or reduced state of the flavin; terms in brackets, enzyme-bound species; kcat, first-order rate constant for substrate transformation and product dissociation; KM, Michaelis−Menten constant; KOx, KSemi, and KRed, dissociation constants, referring to the oxidized, semireduced, and reduced enzyme states, respectively.


Reduction of hydrophilic ubiquinones by the flavin in mitochondrial NADH:ubiquinone oxidoreductase (Complex I) and production of reactive oxygen species.

King MS, Sharpley MS, Hirst J - Biochemistry (2009)

Mechanism of Quinone Reduction at the Flavin Active Site, Incorporating both the Ping-Pong and Ping-Pong-Pong Reactions, and Double-Substrate InhibitionThe ping-pong and ping-pong-pong reactions diverge at [FlRed Q]: the ping-pong reaction follows the dotted arrow to produce QH2 and returns directly to [FlOx], and the ping-pong-pong reaction follows the outside pathway to form two semiquinones (QH•). Abbreviations: FlOx/Red, oxidized or reduced state of the flavin; terms in brackets, enzyme-bound species; kcat, first-order rate constant for substrate transformation and product dissociation; KM, Michaelis−Menten constant; KOx, KSemi, and KRed, dissociation constants, referring to the oxidized, semireduced, and reduced enzyme states, respectively.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651670&req=5

sch1: Mechanism of Quinone Reduction at the Flavin Active Site, Incorporating both the Ping-Pong and Ping-Pong-Pong Reactions, and Double-Substrate InhibitionThe ping-pong and ping-pong-pong reactions diverge at [FlRed Q]: the ping-pong reaction follows the dotted arrow to produce QH2 and returns directly to [FlOx], and the ping-pong-pong reaction follows the outside pathway to form two semiquinones (QH•). Abbreviations: FlOx/Red, oxidized or reduced state of the flavin; terms in brackets, enzyme-bound species; kcat, first-order rate constant for substrate transformation and product dissociation; KM, Michaelis−Menten constant; KOx, KSemi, and KRed, dissociation constants, referring to the oxidized, semireduced, and reduced enzyme states, respectively.
Bottom Line: Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as rotenone and piericidin A).Here, we show that inhibitor-insensitive ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the flavin mononucleotide cofactor in the active site for NADH oxidation.The factors which determine the balance of reactivity between the two sites of ubiquinone reduction (the energy-transducing site and the flavin site) and the implications for mechanistic studies of ubiquinone reduction by complex I are discussed.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.

ABSTRACT
NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound enzyme that contains 45 different subunits, a non-covalently bound flavin mononucleotide, and eight iron-sulfur clusters. The mechanisms of NADH oxidation and intramolecular electron transfer by complex I are gradually being defined, but the mechanism linking ubiquinone reduction to proton translocation remains unknown. Studies of ubiquinone reduction by isolated complex I are problematic because the extremely hydrophobic natural substrate, ubiquinone-10, must be substituted with a relatively hydrophilic analogue (such as ubiquinone-1). Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as rotenone and piericidin A). Here, we show that inhibitor-insensitive ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the flavin mononucleotide cofactor in the active site for NADH oxidation. Moreover, semiquinones produced at the flavin site initiate redox cycling reactions with molecular oxygen, producing superoxide radicals and hydrogen peroxide. The ubiquinone reactant is regenerated, so the NADH:Q reaction becomes superstoichiometric. Idebenone, an artificial ubiquinone showing promise in the treatment of Friedreich's Ataxia, reacts at the flavin site. The factors which determine the balance of reactivity between the two sites of ubiquinone reduction (the energy-transducing site and the flavin site) and the implications for mechanistic studies of ubiquinone reduction by complex I are discussed. Finally, the possibility that the flavin site in complex I catalyzes redox cycling reactions with a wide range of compounds, some of which are important in pharmacology and toxicology, is discussed.

Show MeSH
Related in: MedlinePlus