Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

Patel, S.; Ragan, C I

doi:10.1042/bj2560521

Cited by 16 publications

(12 citation statements)

References 31 publications

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“…It is well accepted that the redox reaction of complex I is accompanied by conformational changes. This was experimenally shown in the mitochondrial complex I from bovine heart by cross-link experiments and treatment with trypsin [22,34,35]. Conformational changes were not detectable when the complex was treated with NADH and K 3 (FeCN) 6 .…”

Section: Resultsmentioning

confidence: 75%

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Pohl

Schneider

Hielscher

et al. 2008

Biochemical Society Transactions

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The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex consisting of a peripheral and a membrane arm. The peripheral arm contains all known cofactors and the NADH-binding site, whereas the membrane arm has to be involved in proton translocation. Owing to this, a conformation-linked mechanism for redox-driven proton translocation is discussed. By means of electron microscopy, we show that both arms of the Escherichia coli complex I are widened after the addition of NADH but not of NADPH. NADH-induced conformational changes were also detected in solution: ATR-FTIR (attenuated total reflection Fourier-transform infrared) of the soluble NADH dehydrogenase fragment of the complex indicates protein re-arrangements induced by the addition of NADH. EPR spectroscopy of surface mutants of the complex containing a covalently bound spin label at distinct positions demonstrates NADH-dependent conformational changes in both arms of the complex.

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Section: Resultsmentioning

confidence: 75%

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Pohl

Schneider

Hielscher

et al. 2008

Biochemical Society Transactions

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“…Cross-linking of Complex I was carried out exactly as described in the preceding paper [2], except that DSST was freshly dissolved in 0.25 M-sucrose/50 mM-triethanolamine/HCl buffer and adjusted to pH 8. Mitochondria were dialysed for 1 h against 200 vol.…”

Section: Cross-linkingmentioning

confidence: 99%

“…In the preceding paper [2], we described the use of cross-linking agents to look at subunit-subunit associations in isolated Complex I. The subunits which were most extensively cross-linked were those constituting the iron-protein domain of the enzyme, and other work with a variety of hydrophilic reagents [3] had suggested that these subunits form a major part of those regions of the enzyme exposed to the aqueous phases on either side of the membrane.…”

Section: Introductionmentioning

confidence: 99%

Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

Patel

Cleeter

Ragan

1988

Biochemical Journal

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The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.

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“…1 and 2), a component of the hydrophobic fraction of the enzyme [8]. Attempts to immuneprecipitate this subunit from SDS-treated extracts with a monospecific antiserum to the bovine 39000Mr subunit [18] were not successful, and definitive identification will require sequencing of this protein.…”

Section: Identification Of the Bovine Mitochondrial Gene Productsmentioning

confidence: 99%

Identification of the subunits of bovine NADH dehydrogenase which are encoded by the mitochondrial genome

Gibb

Ragan

1990

Biochemical Journal

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Products of the mitochondrial genome were identified in the bovine kidney cell line NBL-1 by labelling with [35S]methionine in the presence of cycloheximide. Seven proteins were precipitated by an antiserum to bovine heart NADH dehydrogenase, corresponding to the seven mitochondrial gene products identified in the human HeLa cell line. Comparison of these mitochondrial gene products with purified bovine NADH dehydrogenase by SDS/gel electrophoresis revealed that the ND-5 product is probably a previously unidentified protein of apparent Mr 51,000, and the ND-4 product is the protein of apparent Mr 39,000.

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Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

Cited by 16 publications

References 31 publications

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

Identification of the subunits of bovine NADH dehydrogenase which are encoded by the mitochondrial genome

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