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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.

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NADH is oxidized superstoichiometrically in aerobic quinone-containing solutions. Complex I was incubated in the presence of ∼50 μM Q (dotted lines) in varying concentrations of NADH (from 0 to 200 μM). The NADH concentrations were measured at 2 min intervals, and the amount of NADH oxidized was determined by comparing measured and initial NADH concentrations. (A) NADH oxidation by 50 μM Q1 in an aerobic solution in the presence or absence of rotenone (Rot.) and/or asolectin (Aso.). NADH is oxidized superstoichiometrically. (B) An experiment equivalent to that conducted for panel A but in an anaerobic solution (the symbols are conserved). NADH is not oxidized superstoichiometrically. (C) NADH oxidation by 50 μM DQ in the presence of asolectin, in anaerobic (○) and aerobic (●) solutions. NADH is not oxidized superstoichiometrically. (D) NADH oxidation by 50 μM Q1, IDE, and Q0 in an aerobic solution in the presence of rotenone and asolectin. NADH is oxidized superstoichiometrically. Conditions: 20 mM Tris-HCl, pH 7.55, 32 °C, 2.3 μM rotenone, 0.4 mg/mL asolectin.
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fig4: NADH is oxidized superstoichiometrically in aerobic quinone-containing solutions. Complex I was incubated in the presence of ∼50 μM Q (dotted lines) in varying concentrations of NADH (from 0 to 200 μM). The NADH concentrations were measured at 2 min intervals, and the amount of NADH oxidized was determined by comparing measured and initial NADH concentrations. (A) NADH oxidation by 50 μM Q1 in an aerobic solution in the presence or absence of rotenone (Rot.) and/or asolectin (Aso.). NADH is oxidized superstoichiometrically. (B) An experiment equivalent to that conducted for panel A but in an anaerobic solution (the symbols are conserved). NADH is not oxidized superstoichiometrically. (C) NADH oxidation by 50 μM DQ in the presence of asolectin, in anaerobic (○) and aerobic (●) solutions. NADH is not oxidized superstoichiometrically. (D) NADH oxidation by 50 μM Q1, IDE, and Q0 in an aerobic solution in the presence of rotenone and asolectin. NADH is oxidized superstoichiometrically. Conditions: 20 mM Tris-HCl, pH 7.55, 32 °C, 2.3 μM rotenone, 0.4 mg/mL asolectin.

Mentions: The reaction between NADH and quinone, to form NAD+ and quinol, is a two-electron transfer with a 1:1 stoichiometry, but under conditions which favor quinone reduction at the flavin site, the reaction becomes superstoichiometric (Figure 4). The superstoichiometry of Q1 reduction by complex I has also been noted previously (24,41). Figure 4A shows that 50 μM Q1 is able to oxidize more than 50 μM NADH, whether asolectin and rotenone are present or not. Conditions which favor reaction at the flavin site increase the superstoichiometry, but those that favor the hydrophobic Q-site decrease it. Only quinols are formed at the hydrophobic, physiological Q-site, strongly suggesting that it is semiquinones formed at the flavin site that are responsible for the superstoichiometry, and that the quinols are stable end products. Figure 4B shows that superstoichiometric reactions do not occur unless O2 is present. The rates of NADH oxidation themselves do not depend on the presence of O2, showing that O2 acts indirectly, “recycling” the semiquinone to the quinone, to be reduced again. Figure 4C shows that NADH:DQ oxidoreduction is stoichiometric in both the presence and absence of O2, confirming that the superstoichiometry is associated only with reactions at the flavin site, and Figure 4D compares the reactions of Q1, IDE, and Q0, all of which are superstoichiometric.


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)

NADH is oxidized superstoichiometrically in aerobic quinone-containing solutions. Complex I was incubated in the presence of ∼50 μM Q (dotted lines) in varying concentrations of NADH (from 0 to 200 μM). The NADH concentrations were measured at 2 min intervals, and the amount of NADH oxidized was determined by comparing measured and initial NADH concentrations. (A) NADH oxidation by 50 μM Q1 in an aerobic solution in the presence or absence of rotenone (Rot.) and/or asolectin (Aso.). NADH is oxidized superstoichiometrically. (B) An experiment equivalent to that conducted for panel A but in an anaerobic solution (the symbols are conserved). NADH is not oxidized superstoichiometrically. (C) NADH oxidation by 50 μM DQ in the presence of asolectin, in anaerobic (○) and aerobic (●) solutions. NADH is not oxidized superstoichiometrically. (D) NADH oxidation by 50 μM Q1, IDE, and Q0 in an aerobic solution in the presence of rotenone and asolectin. NADH is oxidized superstoichiometrically. Conditions: 20 mM Tris-HCl, pH 7.55, 32 °C, 2.3 μM rotenone, 0.4 mg/mL asolectin.
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fig4: NADH is oxidized superstoichiometrically in aerobic quinone-containing solutions. Complex I was incubated in the presence of ∼50 μM Q (dotted lines) in varying concentrations of NADH (from 0 to 200 μM). The NADH concentrations were measured at 2 min intervals, and the amount of NADH oxidized was determined by comparing measured and initial NADH concentrations. (A) NADH oxidation by 50 μM Q1 in an aerobic solution in the presence or absence of rotenone (Rot.) and/or asolectin (Aso.). NADH is oxidized superstoichiometrically. (B) An experiment equivalent to that conducted for panel A but in an anaerobic solution (the symbols are conserved). NADH is not oxidized superstoichiometrically. (C) NADH oxidation by 50 μM DQ in the presence of asolectin, in anaerobic (○) and aerobic (●) solutions. NADH is not oxidized superstoichiometrically. (D) NADH oxidation by 50 μM Q1, IDE, and Q0 in an aerobic solution in the presence of rotenone and asolectin. NADH is oxidized superstoichiometrically. Conditions: 20 mM Tris-HCl, pH 7.55, 32 °C, 2.3 μM rotenone, 0.4 mg/mL asolectin.
Mentions: The reaction between NADH and quinone, to form NAD+ and quinol, is a two-electron transfer with a 1:1 stoichiometry, but under conditions which favor quinone reduction at the flavin site, the reaction becomes superstoichiometric (Figure 4). The superstoichiometry of Q1 reduction by complex I has also been noted previously (24,41). Figure 4A shows that 50 μM Q1 is able to oxidize more than 50 μM NADH, whether asolectin and rotenone are present or not. Conditions which favor reaction at the flavin site increase the superstoichiometry, but those that favor the hydrophobic Q-site decrease it. Only quinols are formed at the hydrophobic, physiological Q-site, strongly suggesting that it is semiquinones formed at the flavin site that are responsible for the superstoichiometry, and that the quinols are stable end products. Figure 4B shows that superstoichiometric reactions do not occur unless O2 is present. The rates of NADH oxidation themselves do not depend on the presence of O2, showing that O2 acts indirectly, “recycling” the semiquinone to the quinone, to be reduced again. Figure 4C shows that NADH:DQ oxidoreduction is stoichiometric in both the presence and absence of O2, confirming that the superstoichiometry is associated only with reactions at the flavin site, and Figure 4D compares the reactions of Q1, IDE, and Q0, all of which are superstoichiometric.

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