Abstract:Biochemical characterization of the inhibition mechanism of Deltalac-acetogenins synthesized in our laboratory indicated that they are a new type of inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) [Murai, M., et al. (2006) Biochemistry 45, 9778-9787]. To identify the binding site of Deltalac-acetogenins with a photoaffinity labeling technique, we synthesized a photoreactive Deltalac-acetogenin ([(125)I]diazinylated Deltalac-acetogenin, [(125)I]DAA) which has a small photoreac… Show more
“…Since NBD-Acg, which binds Complex I in almost equimolar proportion, should reside like acetogenin, the conformational changes can be localized between the hydrophobic and hydrophilic domains, in accordance with the data obtained by acetogenin photoaffinity labeling of mitochondrial Complex I [41, 42, 43, 44]. Two fragments of the ND1 (NuoH in E. coli , Nqo8 in T. thermophilus ) subunit were labeled by acetogenin.…”
Section: Discussionsupporting
confidence: 84%
“…3). The acetogenin moiety was reported to bind Complex I between the hydrophobic and hydrophilic domains in the tight vicinity of the ubiquinone channel outlet [41, 42, 43, 44]; fluorescence of NBD is highly sensitive to the hydrophobicity of its environment, thus it should indicate structural re-arrangements in this area.Fig. 2Structure of a non-natural acetogenin stereoisomer ( 1 ), a related NBD (nitrobenzoxadiazole) analog (NBD-Acg, 2 ) and the Annine 6+ dye ( 3 ).Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Two fragments of the ND1 (NuoH in E. coli , Nqo8 in T. thermophilus ) subunit were labeled by acetogenin. One of them composes TMS 4th and 5th and part of the loop between 5th and 6th TMSs (site A) and the other includes 6th TMS and the second part of the loop between 5th and 6th TMSs (site B) [42, 44]. Acetogenin binding to the site B was suppressed by exogenous short-chain ubiquinone [42] suggesting a proximity of an entrance for pool ubiquinone.…”
Section: Discussionmentioning
confidence: 99%
“…One of them composes TMS 4th and 5th and part of the loop between 5th and 6th TMSs (site A) and the other includes 6th TMS and the second part of the loop between 5th and 6th TMSs (site B) [42, 44]. Acetogenin binding to the site B was suppressed by exogenous short-chain ubiquinone [42] suggesting a proximity of an entrance for pool ubiquinone. Recent studies of the acetogenin-binding site in bovine Complex I also mapped Asp160 in the 49 kDa subunit (Asp139 in Nqo4 in T. thermophilus, Asp 329 in NuoCD in E. coli ) [43] which is located in the putative quinone binding cavity [2].…”
Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.
“…Since NBD-Acg, which binds Complex I in almost equimolar proportion, should reside like acetogenin, the conformational changes can be localized between the hydrophobic and hydrophilic domains, in accordance with the data obtained by acetogenin photoaffinity labeling of mitochondrial Complex I [41, 42, 43, 44]. Two fragments of the ND1 (NuoH in E. coli , Nqo8 in T. thermophilus ) subunit were labeled by acetogenin.…”
Section: Discussionsupporting
confidence: 84%
“…3). The acetogenin moiety was reported to bind Complex I between the hydrophobic and hydrophilic domains in the tight vicinity of the ubiquinone channel outlet [41, 42, 43, 44]; fluorescence of NBD is highly sensitive to the hydrophobicity of its environment, thus it should indicate structural re-arrangements in this area.Fig. 2Structure of a non-natural acetogenin stereoisomer ( 1 ), a related NBD (nitrobenzoxadiazole) analog (NBD-Acg, 2 ) and the Annine 6+ dye ( 3 ).Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Two fragments of the ND1 (NuoH in E. coli , Nqo8 in T. thermophilus ) subunit were labeled by acetogenin. One of them composes TMS 4th and 5th and part of the loop between 5th and 6th TMSs (site A) and the other includes 6th TMS and the second part of the loop between 5th and 6th TMSs (site B) [42, 44]. Acetogenin binding to the site B was suppressed by exogenous short-chain ubiquinone [42] suggesting a proximity of an entrance for pool ubiquinone.…”
Section: Discussionmentioning
confidence: 99%
“…One of them composes TMS 4th and 5th and part of the loop between 5th and 6th TMSs (site A) and the other includes 6th TMS and the second part of the loop between 5th and 6th TMSs (site B) [42, 44]. Acetogenin binding to the site B was suppressed by exogenous short-chain ubiquinone [42] suggesting a proximity of an entrance for pool ubiquinone. Recent studies of the acetogenin-binding site in bovine Complex I also mapped Asp160 in the 49 kDa subunit (Asp139 in Nqo4 in T. thermophilus, Asp 329 in NuoCD in E. coli ) [43] which is located in the putative quinone binding cavity [2].…”
Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.
“…33 Clearly, the contribution of at least four distinct subunits to the large Q-binding pocket would be sufficient to account for the likely presence of reaction sites for the quinone substrate, its semiquinone intermediate and/or the quinol product. 2, 6, 36, 37 …”
Section: Potent Complex I Inhibitors May Physically Block Its Conformmentioning
Mitochondrial dysfunction often leads to cell death and disease. We can now draw correlations between the dysfunction of one of the most important mitochondrial enzymes, NADH:ubiquinone reductase or complex I, and its structural organization thanks to the recent advances in the X-ray structure of its bacterial homologs. The new structural information on bacterial complex I provide essential clues to finally understand how complex I may work. However, the same information remains difficult to interpret for many scientists working on mitochondrial complex I from different angles, especially in the field of cell death. Here, we present a novel way of interpreting the bacterial structural information in accessible terms. On the basis of the analogy to semi-automatic shotguns, we propose a novel functional model that incorporates recent structural information with previous evidence derived from studies on mitochondrial diseases, as well as functional bioenergetics.
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