Role of the Isoprenyl Tail of Ubiquinone in Reaction with Respiratory Enzymes:  Studies with Bovine Heart Mitochondrial Complex I and Escherichia coli bo-Type Ubiquinol Oxidase

Sakamoto, Kimitoshi; Miyoshi, Hideto; Ohshima, Michiyo; Kuwabara, Kaoru; Kano, Kenji; Akagı, Takanori; Mogi, Tatsushi; Iwamura, Hajime

doi:10.1021/bi981193u

Cited by 42 publications

(30 citation statements)

References 29 publications

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“…It is possible that the binding of IDE is enhanced by hydrogen bonding between its terminal hydroxyl group and a nearby charged residue in the site. For the hydrophobic site, Sakamoto and co-workers studied the reduction of a range of coenzyme Q 2 derivatives by complex I (53). They concluded that the role of the tail is not simply to enhance the hydrophobicity and attributed the high affinity of Q 2 itself to molecular interactions between the binding site and the methyl branch and π-system of the first isoprene unit.…”

Section: Discussionmentioning

confidence: 99%

Reduction of Hydrophilic Ubiquinones by the Flavin in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) and Production of Reactive Oxygen Species

2009

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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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Section: Discussionmentioning

confidence: 99%

Reduction of Hydrophilic Ubiquinones by the Flavin in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) and Production of Reactive Oxygen Species

2009

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“…Low efficiencies per se do not necessarily dispute the occurrence of catalytic reduction at the quinone reaction site because (i) the apparent electron transfer efficiency of hydrophobic UQ 4 is also low in SMPs, and (ii) the efficiencies of UQ 2 and decyl-UQ (an alkyl derivative of UQ 2 ) markedly vary even with slight structural modifications in the side chain as well as the quinone head-ring (42,43). Therefore, the result that their head-rings reached the reaction site is difficult to reconcile with the channel models.…”

Section: Quinone/inhibitor-binding Pocket In Respiratory Complex Imentioning

confidence: 99%

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches

Uno¹,

Kimura²,

Murai

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma Banerjee NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models. Figure 2. Structures of photoreactive amilorides synthesized in this study. PRA1 and PRA2 used in our previous work (17) are also shown. A photolabile azido group attached to each compound is indicated by a gray circle. The concentration in parentheses is the average IC 50 value, which is the molar concentration (nanomolar) needed to reduce the control NADH oxidase activity in bovine heart SMPs (30 g of proteins/ml) by 50%. As a reference, the IC 50 value of bullatacin was 1.1 (Ϯ 0.09) nM under the same experimental conditions. Quinone/inhibitor-binding pocket in respiratory complex I

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“…The effects of various inhibitors on the steady state kinetics of the enzyme have provided further evidence that there must be more than one Q-binding site [11, 13, 14], and the data have been interpreted in terms of two sites (Q L or Q H ) with the inhibitors binding to one or the other Q-binding sites. The reactivity of the enzyme with synthetic quinone analogues has also been examined, showing the roles of the ring substituents and the hydrophobic side chain in determining the kinetic parameters [15, 16]. …”

Section: Introductionmentioning

confidence: 99%

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Yap

Lin

Ouyang

et al. 2010

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Summary Cytochrome bo3 is the major respiratory oxidase located in the cytoplasmic membrane of E. coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (QH). The quinone bound at the QH site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the QH site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1-10, do not displace ubiquinone-8 from the QH site. The data clearly support the existence of a second binding site for ubiquinone, the QL site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1-10 may compete for binding with quinol at the QL site while, at the same time, preventing formation of the ubisemiquinone at the QH site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.

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Role of the Isoprenyl Tail of Ubiquinone in Reaction with Respiratory Enzymes: Studies with Bovine Heart Mitochondrial Complex I and Escherichia coli bo-Type Ubiquinol Oxidase

Cited by 42 publications

References 29 publications

Reduction of Hydrophilic Ubiquinones by the Flavin in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) and Production of Reactive Oxygen Species

Reduction of Hydrophilic Ubiquinones by the Flavin in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) and Production of Reactive Oxygen Species

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

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