Purification of three iron-sulfur proteins from the iron-protein fragment of mitochondrial NADH-ubiquinone oxidoreductase

Ci, Ragan; Ym, Galante; Hatefi, Youssef

doi:10.1021/bi00539a035

Cited by 58 publications

(20 citation statements)

References 23 publications

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“…Treatment of bovine Complex I with perchlorate leads to the breakup of the complex into three fragments: a water-soluble flavoprotein fragment (FP), a water-soluble iron protein (IP), and a hydrophobic protein fraction (HP) (45,(101)(102)(103). The flavoprotein fragment contains the FMN, an Fe 2 S 2 cluster, and an Fe 4 S 4 cluster.…”

Section: Subunit Description and Localization Of Prosthetic Groupsmentioning

confidence: 99%

Structures and Proton-Pumping Strategies of Mitochondrial Respiratory Enzymes

Schultz

Chan

2001

Annu. Rev. Biophys. Biomol. Struct.

248

177

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Key Words crystal structures, free energy transduction, mitochondrion, redox linkage, respiratory electron transport chain s Abstract Enzymes of the mitochondrial respiratory chain serve as proton pumps, using the energy made available from electron transfer reactions to transport protons across the inner mitochondrial membrane and create an electrochemical gradient used for the production of ATP. The ATP synthase enzyme is reversible and can also serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each of the respiratory enzymes uses a different strategy for performing proton pumping. In this work, the strategies are described and the structural bases for the action of these proteins are discussed in light of recent crystal structures of several respiratory enzymes. The mechanisms and efficiency of proton translocation are also analyzed in terms of the thermodynamics of the substrate transformations catalyzed by these enzymes. CONTENTS

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Section: Subunit Description and Localization Of Prosthetic Groupsmentioning

confidence: 99%

Structures and Proton-Pumping Strategies of Mitochondrial Respiratory Enzymes

Schultz

Chan

2001

Annu. Rev. Biophys. Biomol. Struct.

248

177

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“…The enzyme is composed of Ն43 subunits (1,2), and contains one FMN and 5 EPR-visible iron-sulfur clusters, designated centers N1a, N1b, N2, N3, and N4 (3). Chemical analysis of iron and sulfide suggested the possible presence of 8 iron-sulfur clusters per FMN (4). These results agreed with more recent data on the amino acid sequences of complex I subunits, in which 8 cysteine motifs were found for ligating 6 tetranuclear and 2 binuclear iron-sulfur clusters (1,2).…”

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confidence: 99%

Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

Yamaguchi

Belogrudov²,

Hatefi

1998

Journal of Biological Chemistry

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It has been shown that treatment of bovine mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576) also indicated that complex I undergoes extensive conformation changes when reduced by substrate. Furthermore, we had previously shown that in submitochondrial particles the affinity of complex I for NAD increases by >20-fold in electron transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve protein conformation changes as a key step.In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.Among the enzyme complexes of the mitochondrial electron transport/oxidative phosphorylation system, complex I (NADH-ubiquinone oxidoreductase) has the most complicated structure and mechanism of action. The enzyme is composed of Ն43 subunits (1, 2), and contains one FMN and 5 EPR-visible iron-sulfur clusters, designated centers N1a, N1b, N2, N3, and N4 (3). Chemical analysis of iron and sulfide suggested the possible presence of 8 iron-sulfur clusters per FMN (4). These results agreed with more recent data on the amino acid sequences of complex I subunits, in which 8 cysteine motifs were found for ligating 6 tetranuclear and 2 binuclear iron-sulfur clusters (1, 2).Early studies showed that bovine complex I could be resolved into three domains, designated as flavoprotein (FP), 1 iron-sulfur protein (IP), and hydrophobic protein (HP) (5, 6). This tridomain architecture appears to be the structural design of complex I from Neurospora crassa mitochondria (Ն35 subunits) (7), Escherichia coli plasma membrane (13 subunits) (8, 9), and possibly Paracoccus denitrificans (14 subunits) (10). Bovine FP is water-soluble; it is composed of three subunits with molecular masses of 51, 24, and 9 kDa, and it contains per mol 1 FMN, a tetranuclear iron-sulfur cluster, and a binuclear iron-sulfur cluster. Data from the bovine (11), the Paracoccus (12), and the E. coli (13) enzymes have indicated that the 51-kDa subunit binds NAD(H) and contains FMN and a tetranuclear cluster (center N3), and the 24-kDa s...

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“…It also contains approximately eight Fe-S clusters associated with the 75-, 51-, 24-, and 23-kD proteins and possibly two additional subunits (45)(46)(47). At least two of these appeared reduced in the patient, namely the 51-and 24-kD proteins.…”

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Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins.

Hall¹,

Henriksson²,

Lewis³

et al. 1993

J. Clin. Invest.

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Recently, we described a patient with severe exercise intolerance and episodic myoglobinuria, associated with marked impairment of succinate oxidation and deficient activity of succinate dehydrogenase and aconitase in muscle mitochondria ( 1 ). We now report additional enzymatic and immunological characterization of mitochondria. In addition to severe deficiency of complex II, manifested by reduction of succinate dehydrogenase and succinate:coenzyme Q oxidoreductase activities to 12 and 22% of normal, respectively, complex III activity was reduced to 37% and rhodanese to 48% of normal. Furthermore, although complex I activity was not measured, immunoblot analysis of complex I showed deficiency of the 39-, 24-, 13-, and 9-kD peptides with lesser reductions of the 51-and 18-kD peptides. Immunoblots of complex III showed markedly reduced levels of the mature Rieske protein in mitochondria and elevated levels of its precursor in the cytosol, suggesting deficient uptake into mitochondria. Immunoreactive aconitase was also low. These data, together with the previous documentation of low amounts of the 30-kD iron-sulfur protein and the 13.5-kD subunit of complex II, compared to near normal levels of the 70-kD protein suggest a more generalized abnormality of the synthesis, import, processing, or assembly of a group of proteins containing iron-sulfur clusters. (J. Clin. Invest. 1993. 92:2660-2666.) Key words: myoglobinuria -electron transport * nicotinamide adenine dinucleotide (reduced form) dehydrogenase * ubiquinol-cytochrome c reductase * thiosulfate sulfurtransferase

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Purification of three iron-sulfur proteins from the iron-protein fragment of mitochondrial NADH-ubiquinone oxidoreductase

Cited by 58 publications

References 23 publications

Structures and Proton-Pumping Strategies of Mitochondrial Respiratory Enzymes

Structures and Proton-Pumping Strategies of Mitochondrial Respiratory Enzymes

Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins.

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