Isolation and Characterisation of Subcomplexes of the Mitochondrial NADH: Ubiquinone Oxidoreductase (Complex I)

Finel, Moshe; Majander, Anna; Tyynelä, Jaana; Jong, A.M.P. de; Albracht, S.P.J.; Wikström, M

doi:10.1111/j.1432-1033.1994.tb20046.x

Cited by 61 publications

(38 citation statements)

References 39 publications

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“…These polypeptides were the 49-kDa, TYKY, B17.2, B15, and B14 subunits, which together contain 41 tyrosine residues. Two of these subunits, 49 kDa and TYKY, contain prosthetic groups and/or have been shown to be involved in the enzyme's function (53). However, by Western analysis, the observed levels of modification of these two subunits together are low and cannot account for the loss of function.…”

Section: Discussionmentioning

confidence: 99%

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Murray

Taylor²,

Zhang³

et al. 2003

Journal of Biological Chemistry

303

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There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50 -80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the ␤ and the d subunits of the F 1 F 0 -ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr 122 , while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr 46 , Tyr 50 , and Tyr 51 . In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I sub-

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Section: Discussionmentioning

confidence: 99%

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Murray

Taylor²,

Zhang³

et al. 2003

Journal of Biological Chemistry

303

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“…Subcomplex 1, which corresponds to the peripheral arm, contains 15 subunits and all the known redox cofactors and the NADH binding site. Subcomplex I␣ is subcomplex I plus nine additional subunits (4,(7)(8)(9)(10)(11). Subcomplexes I␤ (13 subunits) and I␥ (6 subunits) provide most of the rest of the membrane arm (4, 8 -12).…”

mentioning

confidence: 99%

Bovine Complex I Is a Complex of 45 Different Subunits

Carroll

Fearnley

Skehel³

et al. 2006

Journal of Biological Chemistry

432

313

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Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex I␤, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.

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“…These three proteins have homologs in the fungus N. crassa, namely the 21.3c-kDa (Duarte et al 1996), 19.3-kDa (Sousa et al 1999, and 51-kDa (Preis et al 1991) subunits, respectively, and are highly conserved from bacteria to mammals. They belong to the peripheral domain of complex I in N. crassa (Videira 1998) or to the IlS subcomplex in bovine (Finel et al 1994). The amino acid sequences of the 21.3c-and 19.3-kDa proteins display consensus motives for binding of the two [4Fe-4S] clusters N6a and N6b (Rasmussen et al 2001) and of the [4Fe-4S] cluster N2 (Duarte et al 2002), respectively.…”

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confidence: 99%

Neurospora Strains Harboring Mitochondrial Disease-Associated Mutations in Iron-Sulfur Subunits of Complex I

et al. 2005

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We subjected the genes encoding the 19.3-, 21.3c-, and 51-kDa iron-sulfur subunits of respiratory chain complex I from Neurospora crassa to site-directed mutagenesis to mimic mutations in human complex I subunits associated with mitochondrial diseases. The V135M substitution was introduced into the 19.3-kDa cDNA, the P88L and R111H substitutions were separately introduced into the 21.3c-kDa cDNA, and the A353V and T435M alterations were separately introduced into the 51-kDa cDNA. The altered cDNAs were expressed in the corresponding null-mutants under the control of a heterologous promoter. With the exception of the A353V polypeptide, all mutated subunits were able to promote assembly of a functional complex I, rescuing the phenotypes of the respective null-mutants. Complex I from these strains displays spectroscopic and enzymatic properties similar to those observed in the wild-type strain. A decrease in total complex I amounts may be the major impact of the mutations, although expression levels of mutant genes from the heterologous promoter were sometimes lower and may also account for complex I levels. We discuss these findings in relation to the involvement of complex I deficiencies in mitochondrial disease.

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Isolation and Characterisation of Subcomplexes of the Mitochondrial NADH: Ubiquinone Oxidoreductase (Complex I)

Cited by 61 publications

References 39 publications

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Bovine Complex I Is a Complex of 45 Different Subunits

Neurospora Strains Harboring Mitochondrial Disease-Associated Mutations in Iron-Sulfur Subunits of Complex I

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