Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function
2020; Elsevier BV; Volume: 295; Issue: 36 Linguagem: Inglês
10.1074/jbc.ra120.014229
ISSN1083-351X
AutoresTakahiro Masuya, Yuki Sano, Hinako Tanaka, Nicole Butler, Takeshi Ito, Tatsuhiko Tosaki, Joel E. Morgan, Masatoshi Murai, Blanca Barquera, Hideto Miyoshi,
Tópico(s)ATP Synthase and ATPases Research
ResumoThe Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR. The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR. The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is the first enzyme in the respiratory chain of many marine and pathogenic bacteria, such as Vibrio alginolyticus, Vibrio cholerae, and Hemophilus influenzae (1Hayashi M. Nakayama Y. Unemoto T. Recent progress in the Na+-translocating NADH-quinone reductase from the marine Vibrio alginolyticus.Biochim. Biophys. Acta. 2001; 1505 (11248187): 37-4410.1016/S0005-2728(00)00275-9Crossref PubMed Scopus (79) Google Scholar, 2Juárez O. Barquera B. Insights into the mechanism of electron transfer and sodium translocation of the Na+-pumping NADH-quinone oxidoreductase.Biochim. Biophys. Acta. 2012; 1817 (22465856): 1823-183210.1016/j.bbabio.2012.03.017Crossref PubMed Scopus (47) Google Scholar). This enzyme couples electron transfer from NADH to UQ with Na+-pumping, generating an electrochemical Na+ gradient across the inner bacterial membrane. Na+-NQR is an integral membrane protein complex that consists of six subunits (NqrA–F) encoded by the nqr operon (2Juárez O. Barquera B. Insights into the mechanism of electron transfer and sodium translocation of the Na+-pumping NADH-quinone oxidoreductase.Biochim. Biophys. Acta. 2012; 1817 (22465856): 1823-183210.1016/j.bbabio.2012.03.017Crossref PubMed Scopus (47) Google Scholar). All of the subunits have transmembrane helices except for NqrA, which is peripherally associated to the core assembly of membrane-spanning subunits, from the cytoplasmic side. Studies using a variety of techniques have defined the locations and redox properties of the cofactors in the enzyme (3Barquera B. Zhou W. Morgan J.E. Gennis R.B. Riboflavin is a component of the Na+-pumping NADH–quinone oxidoreductase from Vibrio cholerae.Proc. Natl. Acad. Sci. U.S.A. 2002; 99 (12122213): 10322-1032410.1073/pnas.162361299Crossref PubMed Scopus (63) Google Scholar, 4Juárez O. Morgan J.E. Barquera B. The electron transfer pathway of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2009; 284 (19155212): 8963-897210.1074/jbc.M809395200Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Juárez O. Morgan J.E. Nilges M.J. Barquera B. Energy transducing redox steps of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20616050): 12505-1251010.1073/pnas.1002866107Crossref PubMed Scopus (44) Google Scholar, 6Bogachev A.V. Bloch D.A. Bertsova Y.V. Verkhovsky M.I. Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase: 2. study of the enzyme by optical spectroscopy.Biochemistry. 2009; 48 (19496622): 6299-630410.1021/bi900525vCrossref PubMed Scopus (33) Google Scholar, 7Casutt M.S. Nedielkov R. Wendelspiess S. Vossler S. Gerken U. Murai M. Miyoshi H. Möller H. Steuber J. Localization of UQ-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2011; 286 (21885438): 40075-4008210.1074/jbc.M111.224980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Juárez O. Neehaul Y. Turk E. Chahboun N. DeMicco J.M. Hellwig P. Barquera B. The role of glycine residues 140 and 141 of subunit B in the functional UQ binding site of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2012; 287 (22645140): 25678-2568510.1074/jbc.M112.366088Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 9Nedielkov R. Steffen W. Steuber J. Möller H.M. NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2013; 288 (24003222): 30597-3060610.1074/jbc.M112.435750Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 10Strickland M. Juárez O. Neehaul Y. Cook D.A. Barquera B. Hellwig P. The conformational changes induced by UQ binding in the Na+-pumping NADH:UQ oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit.J. Biol. Chem. 2014; 289 (25006248): 23723-2373310.1074/jbc.M114.574640Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 11Tuz K. Mezic K.G. Xu T. Barquera B. Juárez O. The kinetic reaction mechanism of the Vibrio cholerae sodium-dependent NADH dehydrogenase.J. Biol. Chem. 2015; 290 (26004776): 20009-2002110.1074/jbc.M115.658773Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 12Belevich N.P. Bertsova Y.V. Verkhovskaya M.L. Baykov A.A. Bogachev A.V. Identification of the coupling step in Na+-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer.Biochim. Biophys. Acta. 2016; 1857 (26655930): 141-14910.1016/j.bbabio.2015.12.001Crossref PubMed Scopus (11) Google Scholar) and led to the consensus that the electron transfer takes place along a pathway consistent of at least five redox cofactors: from NADH to a FAD, a 2Fe-2S center, two covalently bound FMNs, and a riboflavin, before finally reaching UQ. The mechanism responsible for Na+-pumping driven by electron transfer remains largely elusive. There is an X-ray crystallographic model of V. cholerae Na+-NQR, in an oxidized state, with no bound UQ or inhibitor, which has provided valuable information about the overall structure of the enzyme (13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar). However, the model is difficult to reconcile with some of the results obtained in earlier biochemical/biophysical studies (5Juárez O. Morgan J.E. Nilges M.J. Barquera B. Energy transducing redox steps of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20616050): 12505-1251010.1073/pnas.1002866107Crossref PubMed Scopus (44) Google Scholar, 11Tuz K. Mezic K.G. Xu T. Barquera B. Juárez O. The kinetic reaction mechanism of the Vibrio cholerae sodium-dependent NADH dehydrogenase.J. Biol. Chem. 2015; 290 (26004776): 20009-2002110.1074/jbc.M115.658773Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). For example, whereas the sequence of electron transfers through the cofactors of the enzyme has been experimentally determined, the spatial distances between several pairs of redox cofactors in the crystallographic model (e.g. between FMN and riboflavin in NqrB) are too long (29–32 Å) to support physiologically relevant electron transfer (13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar, 15Page C.C. Moser C.C. Chen X. Dutton P.L. Natural engineering principles of electron tunneling in biological oxidation-reduction.Nature. 1999; 402 (10573417): 47-5210.1038/46972Crossref PubMed Scopus (1437) Google Scholar). Also, the proposed binding position of the UQ head-ring in the NqrA subunit is located ∼20 Å above the cytoplasmic membrane surface, and too far (∼40 Å in a straight line) from its proximal electron donor, riboflavin, which is predicted to lie between NqrB and NqrE. Therefore, as Steuber et al. (13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar) and others have suggested, the subunits harboring the cofactors and the UQ-binding cavity may need to undergo large conformational rearrangements that reduce the spatial gaps at appropriated times during catalytic turnover. In addition, Steuber et al. (13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar) proposed that NqrD and NqrE bind a sixth cofactor, a single iron coordinated by four cysteine residues. This extra cofactor would decrease the distance of one of the longest electron transfers (i.e. from the 2Fe-2S center in NqrF to FMN in NqrC), but does not solve the distance-rate problem entirely. Also the presence of this putative iron center, and its redox function, remain to be verified by further experiments because earlier kinetic and spectroscopic studies, including EPR measurements, have not provided evidence for an additional redox center (6Bogachev A.V. Bloch D.A. Bertsova Y.V. Verkhovsky M.I. Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase: 2. study of the enzyme by optical spectroscopy.Biochemistry. 2009; 48 (19496622): 6299-630410.1021/bi900525vCrossref PubMed Scopus (33) Google Scholar, 16Barquera B. Morgan J.E. Lukoyanov D. Scholes C.P. Gennis R.B. Mark J. Nilges M.J. X- and W-band EPR and Q-band ENDOR studies of the flavin radical in the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae.J. Am. Chem. Soc. 2003; 125 (12515529): 265-27510.1021/ja0207201Crossref PubMed Scopus (69) Google Scholar, 17Bogachev A.V. Kulik L.V. Bloch D.A. Bertsova Y.V. Fadeeva M.S. Verkhovsky M.I. Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase. 1. Electron paramagnetic resonance study of the enzyme.Biochemistry. 2009; 48 (19496621): 6291-629810.1021/bi900524mCrossref PubMed Scopus (31) Google Scholar). We previously discovered that aurachin D-42 (Fig. 1), one of numerous aurachins in a chemical library screened in our laboratory, is a very potent inhibitor of V. cholerae Na+-NQR (14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The inhibitory potency of aurachin D-42 (IC50 value ∼2 nm) is comparable with that of korormicin A and is remarkably greater (∼500-fold) than that of HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide), a commercially available aurachin C-type inhibitor, which has been widely used in earlier Na+-NQR research (6Bogachev A.V. Bloch D.A. Bertsova Y.V. Verkhovsky M.I. Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase: 2. study of the enzyme by optical spectroscopy.Biochemistry. 2009; 48 (19496622): 6299-630410.1021/bi900525vCrossref PubMed Scopus (33) Google Scholar, 7Casutt M.S. Nedielkov R. Wendelspiess S. Vossler S. Gerken U. Murai M. Miyoshi H. Möller H. Steuber J. Localization of UQ-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2011; 286 (21885438): 40075-4008210.1074/jbc.M111.224980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Juárez O. Neehaul Y. Turk E. Chahboun N. DeMicco J.M. Hellwig P. Barquera B. The role of glycine residues 140 and 141 of subunit B in the functional UQ binding site of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2012; 287 (22645140): 25678-2568510.1074/jbc.M112.366088Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 9Nedielkov R. Steffen W. Steuber J. Möller H.M. NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2013; 288 (24003222): 30597-3060610.1074/jbc.M112.435750Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 10Strickland M. Juárez O. Neehaul Y. Cook D.A. Barquera B. Hellwig P. The conformational changes induced by UQ binding in the Na+-pumping NADH:UQ oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit.J. Biol. Chem. 2014; 289 (25006248): 23723-2373310.1074/jbc.M114.574640Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 11Tuz K. Mezic K.G. Xu T. Barquera B. Juárez O. The kinetic reaction mechanism of the Vibrio cholerae sodium-dependent NADH dehydrogenase.J. Biol. Chem. 2015; 290 (26004776): 20009-2002110.1074/jbc.M115.658773Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 12Belevich N.P. Bertsova Y.V. Verkhovskaya M.L. Baykov A.A. Bogachev A.V. Identification of the coupling step in Na+-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer.Biochim. Biophys. Acta. 2016; 1857 (26655930): 141-14910.1016/j.bbabio.2015.12.001Crossref PubMed Scopus (11) Google Scholar). In our previous work, we characterized the binding site for UQ as well as aurachin D-42 in V. cholerae Na+-NQR by means of photoaffinity labeling using synthetic photoreactive derivatives (14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). That study produced three important findings. First, the UQ head-ring labels a part of the rear wall of the UQ binding cavity in the NqrA subunit (NqrA-Leu32−Met39 and Phe131−Lys138, Fig. 2, A and B), which was predicted by the crystallographic model (13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar). Casutt et al. (7Casutt M.S. Nedielkov R. Wendelspiess S. Vossler S. Gerken U. Murai M. Miyoshi H. Möller H. Steuber J. Localization of UQ-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2011; 286 (21885438): 40075-4008210.1074/jbc.M111.224980Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) also identified NqrA as the binding subunit of the UQ head-ring using a different photoreactive UQ probe. Second, the photoreactive aurachin D-type inhibitors ([125I]PAD-1 and [125I]PAD-2, Fig. 1) label the N-terminal region of NqrB (NqrB-Tyr23−Gly89), which protrudes from the membrane and forms a long stretch that anchors NqrA to the transmembrane subunits (Fig. 2C). Note that only the segment NqrB-Gly38−Gly89 is shown in Fig. 2C because the region Met1−Pro37 was not included in the structural model (13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar). Although the region labeled by [125I]PAD-1 and [125I]PAD-2 is in NqrB and the region labeled by the UQ probe is in NqrA, Fig. 2, B and C, shows that the two loci are adjacent, or in close proximity, in the assembled structure. Third, the photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2 rather than being competitively suppressed in the presence of an excess of other inhibitors (including their nonradioactive analogs), is enhanced under some experimental conditions (for example, when a molar ratio of 125I-incorporated inhibitor to Na+-NQR is relatively low). Note that we refer to this seemingly paradoxical result as "unusual competitive behavior" throughout this manuscript.Figure 2X-ray crystallographic model of V. cholerae Na+-NQR and the binding sites of UQ and inhibitors. A, entire structure of V. cholerae Na+-NQR (PDB entry 4P6V). Note that a part of the N-terminal stretch (Met1–Pro37) of NqrB was not modeled in Ref. 13Steuber J. Vohl G. Casutt M.S. Vorburger T. Diederichs K. Fritz G. Structure of the Vcholerae Na+-pumping NADH:quinone oxidoreductase.Nature. 2014; 516 (25471880): 62-6710.1038/nature14003Crossref PubMed Scopus (78) Google Scholar. B, the area marked by a gray square in panel A was expanded. The binding region (red) of the UQ head-ring in NqrA identified in the previous work (14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) is indicated. The putative solvent-accessible cavity is marked by a red oval. C, the binding region of [125I]PAD-1 and [125I]PAD-2 in NqrB (dark brown) identified in the previous work (14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The red circle marks an entrance to the putative binding cavity of the UQ head-ring. D, the binding regions of [125I]PAD-3 (His153−Lys191 in yellow) and [125I]PAD-4/[125I]PKRD-1 (His153−Lys191 and Trp23−Lys54 in dark brown) in NqrB are shown. The loop connecting TMHs 2 − 3 (His153−Gly158) is indicated by yellow spheres. The NqrB-Asp49, -Asp52, -Glu154, and -Glu157 are shown as a red stick model. E, close up view of the binding regions of [125I]PAD-3, [125I]PAD-4, and [125I]PKRD-1 in NqrB. The NqrB-Asp49, -Asp52, -Glu154, and -Glu157 are shown in red. The red circle marks an entrance to the putative binding cavity of the UQ head-ring.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The first and second findings together indicate that the binding positions of the UQ head-ring and aurachin D-type inhibitors are close to each other, but it is not clear whether they actually overlap (Fig. 2, B and C). This notion seems to be consistent with the earlier steady-state kinetic studies, which suggested that neither HQNO nor korormicin A competes directly with UQ1 (9Nedielkov R. Steffen W. Steuber J. Möller H.M. NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae.J. Biol. Chem. 2013; 288 (24003222): 30597-3060610.1074/jbc.M112.435750Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 11Tuz K. Mezic K.G. Xu T. Barquera B. Juárez O. The kinetic reaction mechanism of the Vibrio cholerae sodium-dependent NADH dehydrogenase.J. Biol. Chem. 2015; 290 (26004776): 20009-2002110.1074/jbc.M115.658773Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 18Nakayama Y. Hayashi M. Yoshikawa K. Mochida K. Unemoto T. Inhibitor studies of a new antibiotic korormicin, 2-n-heptyl-4-hydroxyquinoline N-oxide and Ag+ toward the Na+-translocating NADH-quinone reductase from the marine Vibrio alginolyticus.Biol. Pharm. Bull. 1999; 22 (10549856): 1064-106710.1248/bpb.22.1064Crossref PubMed Scopus (36) Google Scholar, 19Yoshikawa K. Nakayama Y. Hayashi M. Unemoto T. Mochida K. Korormicin, an antibiotic specific for Gram-negative marine bacteria, strongly inhibits the respiratory chain-linked Na+-translocating NADH:quinone reductase from the marine Vibrio alginolyticus.J. Antibiot. 1999; 52 (10344574): 182-18510.7164/antibiotics.52.182Crossref PubMed Scopus (37) Google Scholar, 20Hayashi M. Shibata N. Nakayama Y. Yoshikawa K. Unemoto T. Korormicin insensitivity in Vibrio alginolyticus is correlated with a single point mutation of Gly-140 in the NqrB subunit of the Na+-translocating NADH-quinone reductase.Arch. Biochem. Biophys. 2002; 401: 173-17710.1016/S0003-9861(02)00007-3Crossref PubMed Scopus (29) Google Scholar). Although these binding regions protrude from the membrane toward the cytoplasm, we cannot rule out the possibility that the crystallographic structure might differ from that of the native enzyme, as described above. The third finding is difficult to reconcile with a simple scenario in which different inhibitors share a common binding pocket. To explain the unusual competitive behavior of aurachin D-type inhibitors, we earlier proposed an equilibrium model for the binding of the 125I-incorporated inhibitors based on the following three suppositions: (i) there are two distinct inhibitor-bound states (i.e. one-inhibitor– and two-inhibitor–bound states), (ii) the inhibitor binding to the second site is almost negligible unless the first site is already occupied by a molecule of the inhibitor, and (iii) the yield of the labeling reaction in the two-inhibitor–bound state is higher than that in the one-inhibitor-bound state (see Ref. 14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar for details). Although we cannot exclude other scenarios that could explain the unusual competitive behavior, this model accounts for the consecutive changes in the nature of the competition (from enhancement to suppression) as the concentration of competitor increases. Thus, our previous study provided important insights into the terminal electron transfer step in V. cholerae Na+-NQR (14Ito T. Murai M. Ninokura S. Kitazumi Y. Mezic K.G. Cress B.F. Koffas M.A.G. Morgan J.E. Barquera B. Miyoshi H. Identification of the binding sites for UQ and inhibitors in the Na+-pumping NADH-UQ oxidoreductase of Vibrio cholerae by photoaffinity labeling.J. Biol. Chem. 2017; 292 (28298441): 7727-774210.1074/jbc.M117.781393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Nevertheless, to thoroughly elucidate the mechanisms of action of various potent inhibitors of the enzyme, several issues raised by our previous work still need to be addressed, including the following. First, we were unable to examine the inhibition mechanism of korormicin A (Fig. 1), a very potent natural inhibitor of V. cholerae Na+-NQR (19Yoshikawa K. Nakayama Y. Hayashi M. Unemoto T. Mochida K. Korormicin, an antibiotic specific for Gram-negative marine bacteria, strongly inhibits the respiratory chain-linked Na+-translocating NADH:quinone reductase from the marine Vibrio alginolyticus.J. Antibiot. 1999; 52 (10344574): 182-18510.7164/antibiotics.52.182Crossref PubMed Scopus (37) Google Scholar), because no photoreactive derivative was available due to synthetic difficulties related to its complicated structure. Although aurachin-type inhibitors are also able to inhibit other respiratory enzymes such as complex III (21Von Jagow G. Link T.A. 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