Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I
2022; Elsevier BV; Volume: 298; Issue: 7 Linguagem: Inglês
10.1016/j.jbc.2022.102075
ISSN1083-351X
AutoresShinpei Uno, Takahiro Masuya, Oleksii Zdorevskyi, Ryo Ikunishi, Kyoko Shinzawa‐Itoh, Jonathan Lasham, Vivek Sharma, Masatoshi Murai, Hideto Miyoshi,
Tópico(s)Coenzyme Q10 studies and effects
ResumoThe ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQm-1/pUQp-1 and pUQm-2/pUQp-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [125I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [125I]pUQs used, indicating that [125I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model. The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQm-1/pUQp-1 and pUQm-2/pUQp-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [125I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [125I]pUQs used, indicating that [125I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model. Proton-translocating NADH-quinone oxidoreductase (complex I), which is the largest of the respiratory chain enzymes, couples electron transfer from NADH to quinone with the translocation of protons across the membrane. The electrochemical proton gradient produced by complex I drives energy-consuming reactions, such as ATP synthesis via oxidative phosphorylation and substrate transport across the membrane (1Hirst J. Mitochondrial complex I.Annu. Rev. Biochem. 2013; 82: 551-575Crossref PubMed Scopus (395) Google Scholar, 2Sazanov L.A. A giant molecular proton pump: structure and mechanism of respiratory complex I.Nat. Mol. Cell Biol. 2015; 16: 375-388Crossref PubMed Scopus (285) Google Scholar, 3Wirth C. Brandt U. Hunte C. Zickermann V. Structure and function of mitochondrial complex I.Biochim. Biophys. Acta. 2016; 1857: 902-914Crossref PubMed Scopus (182) Google Scholar, 4Wong H.-S. Dighe P.A. Mezera V. Monternier P.-A. Brand M.D. Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions.J. Biol. Chem. 2017; 292: 16804-16809Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The recent rapid advances in single-particle cryo-EM studies (5Zhu J. Vinothkumar K.R. Hirst J. Structure of mammalian respiratory complex I.Nature. 2016; 536: 354-358Crossref PubMed Scopus (342) Google Scholar, 6Blaza J.N. Vinothkumar K.R. Hirst J. Structure of the deactive state of mammalian respiratory complex I.Structure. 2018; 26: 312-319Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 7Fiedorczuk K. Letts J.A. Degliesposti G. Kaszuba K. Skehel M. Sazanov L.A. Atomic structure of the entire mammalian mitochondrial complex I.Nature. 2016; 538: 406-410Crossref PubMed Scopus (319) Google Scholar, 8Wu M. Gu J. Guo R. Huang Y. Yang M. Structure of mammalian respiratory supercomplex I1III2IV1.Cell. 2016; 167: 1598-1609Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 9Agip A.-N.A. Blaza J.N. Gridges H.R. Viscomi C. Rawson S. Muench S.P. et al.Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25: 548-556Crossref PubMed Scopus (127) Google Scholar, 10Guo R. Zong S. Wu M. Gu J. Yang M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2.Cell. 2017; 170: 1247-1257Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 11Parey K. Haapanen O. Sharma V. Köfeler H. Züllig T. Prinz S. et al.High-resolution cryo-EM structures of respiratory complex I: mechanism, assembly, and disease.Sci. Adv. 2019; 5eaax9484Crossref Scopus (69) Google Scholar, 12Grba D.N. Hirst J. Mitochondrial complex I structure reveals ordered water moleculaes for catalysis and proton translocation.Nat. Struct. Mol. Biol. 2020; 27: 892-900Crossref PubMed Scopus (51) Google Scholar, 13Bridges H.R. Fedor J.G. Blaza J.N. Lica A.D. Jussupow A. Jarman O.D. et al.Structure of inhibitor-bound mammalian complex I.Nat. Commun. 2020; 11: 5261Crossref PubMed Scopus (37) Google Scholar, 14Kampjut D. Sazanov L.A. The coupling mechanism of mammalian respiratory complex I.Science. 2020; 370eabc4209Crossref PubMed Google Scholar, 15Parey K. Lasham J. Mills D.J. Djurabekova A. Haapanen O. Yoga E.G. et al.High-resolution structure and dynamics of mitochondrial complex I-insights into the proton pumping mechanism.Sci. Adv. 2021; 7eabj3221Crossref PubMed Scopus (18) Google Scholar) along with computational simulation works (16Sharma V. Belevich G. Gamiz-Hernandez A.P. Róg T. Vattulainen I. Verkhovskaya M.L. et al.Redox-induced activation of the proton pump in the respiratory complex I.Proc. Natl. Acad. Sci. U. S. A. 2015; 122: 11571-11576Crossref Scopus (85) Google Scholar, 17Luca A.D. Gamiz-Hernandez A.P. Kaila V.R.I. Symmetry-related proton transfer pathways in respiratory complex I.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 6314-6321Google Scholar, 18Gamiz-Hernandez A.P. Jussupow A. Johansson M.P. Kaila V.R.I. Terminal electron-proton transfer dynamics in the quinone reduction of respiratory complex I.J. Am. Chem. Soc. 2017; 139: 16282-16288Crossref PubMed Scopus (42) Google Scholar, 19Djurabekova A. Haapanen O. Sharma V. Proton motive function of the terminal antiporter-like subunit in respiratory complex I.Biochim. Biophys. Acta Bioenerg. 2020; 1861: 148185Crossref PubMed Scopus (5) Google Scholar, 20Haapanen O. Sharma V. Redox- and protonation-state driven substrate-protein dynamics in respiratory complex I.Curr. Opin. Electrochem. 2021; 29: 100741Crossref Scopus (7) Google Scholar) provided invaluable information about the structure and functions of the enzyme. These outcomes have led to the consensus that structural and electrostatic rearrangements induced by the quinone reduction, which occurs at the interface between the hydrophilic and membrane arms, transmit to the membrane subunits to trigger proton translocation. Therefore, the quinone reduction is a key part of the energy conversion processes, although the mechanism responsible remains elusive.Structural biology studies identified a long and narrow tunnel-like cavity (∼30 Å long), leading to the suggestion that ubiquinones (UQs) of varying isoprenyl chain lengths enter and transit the cavity to be reduced at the "top" of the channel near the iron–sulfur (Fe–S) cluster N2 and then exit into the membrane (5Zhu J. Vinothkumar K.R. Hirst J. Structure of mammalian respiratory complex I.Nature. 2016; 536: 354-358Crossref PubMed Scopus (342) Google Scholar, 6Blaza J.N. Vinothkumar K.R. Hirst J. Structure of the deactive state of mammalian respiratory complex I.Structure. 2018; 26: 312-319Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 7Fiedorczuk K. Letts J.A. Degliesposti G. Kaszuba K. Skehel M. Sazanov L.A. Atomic structure of the entire mammalian mitochondrial complex I.Nature. 2016; 538: 406-410Crossref PubMed Scopus (319) Google Scholar, 8Wu M. Gu J. Guo R. Huang Y. Yang M. Structure of mammalian respiratory supercomplex I1III2IV1.Cell. 2016; 167: 1598-1609Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 9Agip A.-N.A. Blaza J.N. Gridges H.R. Viscomi C. Rawson S. Muench S.P. et al.Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25: 548-556Crossref PubMed Scopus (127) Google Scholar, 10Guo R. Zong S. Wu M. Gu J. Yang M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2.Cell. 2017; 170: 1247-1257Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 11Parey K. Haapanen O. Sharma V. Köfeler H. Züllig T. Prinz S. et al.High-resolution cryo-EM structures of respiratory complex I: mechanism, assembly, and disease.Sci. Adv. 2019; 5eaax9484Crossref Scopus (69) Google Scholar, 12Grba D.N. Hirst J. Mitochondrial complex I structure reveals ordered water moleculaes for catalysis and proton translocation.Nat. Struct. Mol. Biol. 2020; 27: 892-900Crossref PubMed Scopus (51) Google Scholar, 13Bridges H.R. Fedor J.G. Blaza J.N. Lica A.D. Jussupow A. Jarman O.D. et al.Structure of inhibitor-bound mammalian complex I.Nat. Commun. 2020; 11: 5261Crossref PubMed Scopus (37) Google Scholar, 14Kampjut D. Sazanov L.A. The coupling mechanism of mammalian respiratory complex I.Science. 2020; 370eabc4209Crossref PubMed Google Scholar, 15Parey K. Lasham J. Mills D.J. Djurabekova A. Haapanen O. Yoga E.G. et al.High-resolution structure and dynamics of mitochondrial complex I-insights into the proton pumping mechanism.Sci. Adv. 2021; 7eabj3221Crossref PubMed Scopus (18) Google Scholar). This UQ-accessing tunnel extends from a narrow entry point (∼5 Å diameter), which is located at the middle of the membrane-embedded subunit ND1, to the cluster N2. The so-called quinone-site inhibitors such as piericidin A and rotenone have been considered to block the catalytic reaction of UQ by occupying the tunnel (21Baradaran R. Berrisford J.M. Minhas G.S. Sazanov L.A. Crystal structure of the entire respiratory complex I.Nature. 2013; 494: 443-448Crossref PubMed Scopus (557) Google Scholar, 22Zickermann V. Wirth C. Nasiri H. Siegmund K. Schwalbe H. Hunte C. et al.Mechanistic insight from the crystal structure of mitochondrial complex I.Science. 2015; 347: 44-49Crossref PubMed Scopus (292) Google Scholar, 23Fedor J.G. Jones A.J.Y. Di Luca A. Kaila V.R.I. Hirst J. Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 12737-12742Crossref PubMed Scopus (62) Google Scholar). Recent cryo-EM studies identified the densities attributed to these inhibitors, although two (not one) inhibitor molecules bound inside the tunnel (13Bridges H.R. Fedor J.G. Blaza J.N. Lica A.D. Jussupow A. Jarman O.D. et al.Structure of inhibitor-bound mammalian complex I.Nat. Commun. 2020; 11: 5261Crossref PubMed Scopus (37) Google Scholar, 14Kampjut D. Sazanov L.A. The coupling mechanism of mammalian respiratory complex I.Science. 2020; 370eabc4209Crossref PubMed Google Scholar).In contrast to the rigid and narrow cavity originally modeled in crystallographic maps from Thermus thermophilus complex I (21Baradaran R. Berrisford J.M. Minhas G.S. Sazanov L.A. Crystal structure of the entire respiratory complex I.Nature. 2013; 494: 443-448Crossref PubMed Scopus (557) Google Scholar), recent cryo-EM studies of mammalian complex I indicated that the shape of the UQ-accessing tunnel substantially changes depending on the enzyme's states (e.g., active/deactive or open/closed states) or bound ligands because of large conformational rearrangements of three loops connecting transmembrane helixes (TMHs) 5 to 6 of ND1, TMHs 1 to 2 of ND3, and β1−β2 of 49-kDa subunits (9Agip A.-N.A. Blaza J.N. Gridges H.R. Viscomi C. Rawson S. Muench S.P. et al.Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25: 548-556Crossref PubMed Scopus (127) Google Scholar, 12Grba D.N. Hirst J. Mitochondrial complex I structure reveals ordered water moleculaes for catalysis and proton translocation.Nat. Struct. Mol. Biol. 2020; 27: 892-900Crossref PubMed Scopus (51) Google Scholar, 14Kampjut D. Sazanov L.A. The coupling mechanism of mammalian respiratory complex I.Science. 2020; 370eabc4209Crossref PubMed Google Scholar). Thus, the original idea of a rigid and closed UQ-accessing tunnel based on T. thermophilus complex I (21Baradaran R. Berrisford J.M. Minhas G.S. Sazanov L.A. Crystal structure of the entire respiratory complex I.Nature. 2013; 494: 443-448Crossref PubMed Scopus (557) Google Scholar) has been gradually shifting toward a more flexible one. However, this also raises the following question: if the tunnel architecture changes substantially during catalytic turnover, how are redox reactions of UQ in the tunnel insulated from the protons present in the bulk matrix side (or N phase) of the membrane, because premature protonation of reduced UQ intermediates in the tunnel will result in loss of energy. To explain this, various gating mechanisms have been proposed in recent joint structural–computational studies (15Parey K. Lasham J. Mills D.J. Djurabekova A. Haapanen O. Yoga E.G. et al.High-resolution structure and dynamics of mitochondrial complex I-insights into the proton pumping mechanism.Sci. Adv. 2021; 7eabj3221Crossref PubMed Scopus (18) Google Scholar, 24Yoga E.G. Parey K. Djurabekova A. Haapanen O. Siegmund K. Zwicker K. et al.Essential role of accessory subunit LYRM6 in the mechanism of mitochondrial complex I.Nat. Commun. 2020; 11: 6008Crossref PubMed Scopus (16) Google Scholar).On the other hand, several findings obtained by chemistry-based studies in our laboratory are difficult to reconcile with the UQ-accessing tunnel model (25Uno S. Kimura H. Murai M. Miyoshi H. Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.J. Biol. Chem. 2019; 294: 679-696Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 26Banba A. Tsuji A. Kimura H. Murai M. Miyoshi H. Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I.J. Biol. Chem. 2019; 294: 6550-6561Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 27Uno S. Masuya T. Shinzawa-Itoh K. Lasham J. Haapanen O. Shiba T. et al.Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.J. Biol. Chem. 2020; 295: 2449-2463Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 28Tsuji A. Akao T. Masuya T. Murai M. Miyoshi H. IACS-010759, a potent inhibitor of glycolysis-deficient hypoxic tumor cells, inhibits mitochondrial respiratory complex I through a unique mechanism.J. Biol. Chem. 2020; 295: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 29Masuya T. Uno S. Murai M. Miyoshi H. Pinpoint dual chemical cross-linking explores structural dynamics of the ubiquinone reaction site in mitochondrial complex I.Biochemistry. 2021; 60: 813-824Crossref PubMed Scopus (6) Google Scholar). For example, oversized UQs (OS-UQs), which have an extremely bulky "block" (∼13 Å across) attached to their side chains (e.g., OS-UQ2 and OS-UQ3, Fig. S1), were able to function as electron acceptors from native complex I embedded in bovine heart submitochondrial particles (SMPs) (27Uno S. Masuya T. Shinzawa-Itoh K. Lasham J. Haapanen O. Shiba T. et al.Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.J. Biol. Chem. 2020; 295: 2449-2463Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Molecular dynamics (MD) simulations showed that their transition through the narrow UQ tunnel is not energetically feasible (27Uno S. Masuya T. Shinzawa-Itoh K. Lasham J. Haapanen O. Shiba T. et al.Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.J. Biol. Chem. 2020; 295: 2449-2463Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The reduction of these OS-UQs and proton translocation coupled with their reduction were fully inhibitor sensitive, indicating that the reaction of OS-UQs takes place at the physiological catalytic site in the enzyme. In addition, photoaffinity-labeling studies using various inhibitors showed that they do not necessarily enter the UQ-accessing tunnel but rather bind to different positions around the tunnel (25Uno S. Kimura H. Murai M. Miyoshi H. Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.J. Biol. Chem. 2019; 294: 679-696Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 26Banba A. Tsuji A. Kimura H. Murai M. Miyoshi H. Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I.J. Biol. Chem. 2019; 294: 6550-6561Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 28Tsuji A. Akao T. Masuya T. Murai M. Miyoshi H. IACS-010759, a potent inhibitor of glycolysis-deficient hypoxic tumor cells, inhibits mitochondrial respiratory complex I through a unique mechanism.J. Biol. Chem. 2020; 295: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar).Based on these findings, we proposed that the binding manners of various ligands are more diverse than can be accounted for by the canonical UQ tunnel model. The matrix-side interfacial domain of the 49-kDa, ND1, and PSST subunits, which is peripherally covered by a loop connecting TMHs 1 to 2 of ND3, would be one of the possible areas that bulky ligands can access the UQ reaction cavity (25Uno S. Kimura H. Murai M. Miyoshi H. Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.J. Biol. Chem. 2019; 294: 679-696Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In support of this, we recently demonstrated that Cys39 of ND3 and Asp160 of 49 kDa, which are located on the matrix-side TMHs 1 to 2 loop and deep inside the cavity, respectively, can be crosslinked by synthetic bifunctional crosslinkers (29Masuya T. Uno S. Murai M. Miyoshi H. Pinpoint dual chemical cross-linking explores structural dynamics of the ubiquinone reaction site in mitochondrial complex I.Biochemistry. 2021; 60: 813-824Crossref PubMed Scopus (6) Google Scholar), although the two residues are separated by a channel wall in structural models (6Blaza J.N. Vinothkumar K.R. Hirst J. Structure of the deactive state of mammalian respiratory complex I.Structure. 2018; 26: 312-319Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 7Fiedorczuk K. Letts J.A. Degliesposti G. Kaszuba K. Skehel M. Sazanov L.A. Atomic structure of the entire mammalian mitochondrial complex I.Nature. 2016; 538: 406-410Crossref PubMed Scopus (319) Google Scholar, 9Agip A.-N.A. Blaza J.N. Gridges H.R. Viscomi C. Rawson S. Muench S.P. et al.Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25: 548-556Crossref PubMed Scopus (127) Google Scholar). This finding indicates that the UQ reaction cavity is accessible from the proposed matrix-side domain covered by the ND3-TMHs 1 to 2 loop. Interestingly, an MD simulation study reported that small UQ1 can leave from the UQ reaction cavity to the matrix-side medium through a route, which is also close to the ND3-TMHs 1 to 2 loop (30Haapanen O. Djurabekova A. Sharma V. Role of second quinone binding site in proton pumping by respiratory complex I.Front. Chem. 2019; 7: 221Crossref PubMed Scopus (29) Google Scholar). In addition, a recent cryo-EM study of porcine complex I demonstrated that short-chain UQs such as UQ1 can diffuse into the deep reaction site near the cluster N2 even in the presence of endogenous UQ10 in the tunnel, suggesting that some alternative route exists to support the entry of short-chain UQs into the site (31Gu J. Liu T. Guo R. Zhang L. Yang M. The coupling mechanism of mammalian mitochondrial complex I.Nat. Struct. Mol. Biol. 2022; 29: 172-182Crossref PubMed Scopus (12) Google Scholar).Concerning the reaction of OS-UQs, there remains an important question to be addressed: although OS-UQ2 and OS-UQ3 could function as electron acceptors from the native complex I embedded in SMPs, why were they unable to function with the isolated enzyme? (27Uno S. Masuya T. Shinzawa-Itoh K. Lasham J. Haapanen O. Shiba T. et al.Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.J. Biol. Chem. 2020; 295: 2449-2463Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). To answer this, we tentatively hypothesized that the head-ring of OS-UQs cannot reach the reaction site near the Fe–S cluster N2 because their access route in the native enzyme is altered by detergent solubilizing from the inner mitochondrial membrane (IMM) (27Uno S. Masuya T. Shinzawa-Itoh K. Lasham J. Haapanen O. Shiba T. et al.Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.J. Biol. Chem. 2020; 295: 2449-2463Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) (note that it is unclear whether the access route is the same as the main tunnel identified in the structural studies, and this point is the principal issue to be focused on in this study). Unfortunately, we currently have no direct way of inspecting structural differences, if any, between native and isolated enzymes. To find a clue to the contradiction, further biochemical characterizations of the reaction manners of varying bulky UQs are needed. In particular, it is important to examine whether such different reaction behaviors of OS-UQs between the native and isolated complex I are exceptional cases just for these extremely bulky UQs or if it is also the case for other UQs possessing various chemical blocks in the side chain.With this background, if we are able to produce a pair of UQs satisfying the following two requirements, they may become highly useful chemical tools for investigating the mechanism of UQ reduction as well as the cause of the aforementioned contradictory results. As the first requirement, the pair of UQs must have the same chemical properties except for a subtle difference in widths of their blocks attached to the side chain, namely a pair of narrow and wide UQs. If there are some structural differences in the UQ access route between the native and isolated complex I, as previously hypothesized (27Uno S. Masuya T. Shinzawa-Itoh K. Lasham J. Haapanen O. Shiba T. et al.Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I.J. Biol. Chem. 2020; 295: 2449-2463Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), the obstruction "threshold," which restricts the access of UQs to the deep reaction site near the cluster N2, may also be different between the two enzymes. In that case, the wider UQ may function as an electron acceptor from the native complex I but not from the isolated enzyme because of more severe obstruction. In contrast, the narrower UQ may be free from the obstruction and function with both enzymes. Production of the pair of UQs that satisfies the first requirement would enable us to examine this. Here, similar chemical properties of the UQ pair are advantageous in simplifying comparison of their reaction manners; for example, both UQs exhibit similar partitioning behavior between the reaction medium and IMM. As the second requirement, to identify and compare the binding site of the pair of UQs by a photoaffinity-labeling technique, their side chains must be equipped with both a photolabile group (e.g., an azido group) and detecting tag (e.g., 125I or 3H). Although synthesis of the UQ pair solely satisfying the first requirement may not be particularly difficult, the second requirement highly limits available chemical components for constructing the side chain's framework. The present study was aimed to synthesize a pair of UQs satisfying the two requirements at the same time.Through trial-and-error syntheses, we succeeded in producing two pairs of desired UQs: pUQm-1 and pUQp-1 and their respective hydrophobic analogs pUQm-2 and pUQp-2 (Fig. 1). The wider pUQp-1 and pUQp-2 functioned as efficient electron acceptors from the native complex I but not from the isolated enzyme. Photoaffinity-labeling experiments indicated that the side chains of the four [125I]pUQs predominantly label the ND1 subunit and subsidiarily ND5 and ND2, which are located far from the UQ-accessing tunnel. The labeling profiles against the three subunits varied not only between the narrower and wider UQs but also between the native and isolated complexes. None of the six inhibitors tested, which are considered to occupy the UQ-accessing tunnel (13Bridges H.R. Fedor J.G. Blaza J.N. Lica A.D. Jussupow A. Jarman O.D. et al.Structure of inhibitor-bound mammalian complex I.Nat. Commun. 2020; 11: 5261Crossref PubMed Scopus (37) Google Scholar, 14Kampjut D. Sazanov L.A. The coupling mechanism of mammalian respiratory complex I.Science. 2020; 370eabc4209Crossref PubMed Google Scholar, 22Zickermann V. Wirth C. Nasiri H. Siegmund K. Schwalbe H. Hunte C. et al.Mechanistic insight from the crystal structure of mitochondrial complex I.Science. 2015; 347: 44-49Crossref PubMed Scopus (292) Google Scholar), suppressed all the labeling of ND1 by [125I]pUQs. Based on the results, we discuss the diverse reaction behaviors of UQs in complex I in comparison with the canonical UQ tunnel model. This study presents the first photoaffinity-labeling experiments performed using UQ derivatives with complex I.ResultsMolecular design of photoreactive pUQsTo produce a pair of UQs satisfying the aforementioned two requirements, we synthesized many UQs that have various functionalized benzenes in their side chains. Among them, we obtained two pairs of desired UQs: pUQm-1 and pUQp-1 and their respective hydrophobic derivatives pUQm-2 and pUQp-2 (Fig. 1). The synthetic procedures of these pUQs are described in the supporting information (Schemes S1 and S2). In these pUQs, CF3-diazirine and 125I were used as a photolabile group and detecting tag, respectively. The functionalized benzene part in these pUQs serves not only as the "block" that influences their reaction with the enzyme but also as the essential outfit for photoaffinity labeling. These blocks are significantly less bulky compared with that attached to OS-UQs previously studied (Fig. S1). The chemical properties are the same between pUQm-1 and pUQp-1 and between pUQm-2 and pUQp-2 except for a subtle difference in the width of the benzene part, which was fine-tuned by meta- versus para-substitution patterns of CF3-diazirine and iodine (Fig. 1). The planar molecular width of the para-substituted benzene (pUQp-1 and pUQp-2) is slightly longer (∼1.0 Å) than that of the meta-substituted benzene (pUQm-1 and pUQm-2). Therefore, if these artificial UQs encounter steric obstruction during accessing the reaction site in the enzyme, the para derivatives may be subject to more severe obstruction than the corresponding meta derivatives. Although the difference in their widths is small, it actually served as a key structural factor that differentiates the reaction behaviors between the wider (para) and narrower (meta) pUQs with the isolated complex I, as described hereafter.Modeling and simulations of the binding of pUQs to complex IEarlier chemical biology studies (25Uno S. Kimura H. Murai M. Miyoshi H. Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.J. Biol. Chem. 2019; 294: 679-696Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 26Banba A. Tsuji A. Kimura H. Murai M. Miyoshi H. Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I.J. Biol. Chem. 2019; 294: 6550-6561Abstract F
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