Catalytic Sector of Complex I (NADH:Ubiquinone Oxidoreductase):Subunit Stoichiometry and Substrate-Induced Conformation Changes

Belogrudov, Grigory I.; Hatefi, Youssef

doi:10.1021/bi00181a018

Cited by 112 publications

(76 citation statements)

References 38 publications

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“…Our observations from cross-linking and limited proteolysis experiments are in agreement with previous studies on bovine complex I, where the number of cross-links between hydrophilic subunits was reduced significantly in the presence of NADH or NADPH, but not NAD ϩ (23). The susceptibility of a number of hydrophilic subunits to tryptic degradation was also increased in the presence of NAD(P)H, but not NAD ϩ (26).…”

Section: Discussionsupporting

confidence: 82%

“…In the second model, the catalytic module with electron carriers is distinct from the proton-pumping module. The two modules can be spatially separated; energy transduction takes place through long range conformational changes (4,(21)(22)(23). Sequence comparisons have suggested that subunits NuoL, -M, and -N have evolved from a common ancestor related to K ϩ or Na ϩ /H ϩ antiporters and, thus, are likely to be involved in proton translocation (1,24,25).…”

mentioning

confidence: 99%

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Substrate-induced Conformational Change in Bacterial Complex I

Mamedova

Holt

Carroll

et al. 2004

Journal of Biological Chemistry

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The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes. Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy. When a zerolength cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD ؉ , prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD. Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key ironsulfur cluster N2, is the most mobile subunit. The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG. These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits. Single particle analysis showed the nature of this conformational change. The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also.

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Section: Discussionsupporting

confidence: 82%

mentioning

confidence: 99%

Substrate-induced Conformational Change in Bacterial Complex I

Mamedova

Holt

Carroll

et al. 2004

Journal of Biological Chemistry

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“…In more recent models combinations of direct and conformational coupling mechanisms have been suggested to account for the observed pumping stoichimetry of complex I (7,8). In fact, biochemical (28) and structural evidence (29) for redoxlinked conformational changes of complex I has been presented. However, the spatial separation of all redox centers from the membrane domain (3) seems to exclude any direct link between redox chemistry and vectorial proton translocation as defined by Peter Mitchell's "redox-loop" concept (30).…”

Section: Discussionmentioning

confidence: 99%

The Redox-Bohr Group Associated with Iron-Sulfur Cluster N2 of Complex I

Zwicker¹,

Galkin²,

Dröse

et al. 2006

Journal of Biological Chemistry

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Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near ironsulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redoxBohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H ؉ /2e ؊ . This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.In mammalian cells mitochondrial complex I (NADH: ubiquinone oxidoreductase) provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme (1) cause numerous human disorders (2). Very recently, the x-ray structure of the peripheral arm of complex I from Thermus thermophilus has been solved (3). However, it is still unclear how complex I couples electron transfer from NADH to ubiquinone with the vectorial translocation of four protons across the bioenergetic membrane (4, 5). Numerous hypothetical mechanisms have been proposed for this energy conversion over the years (6 -9). None of the known redox groups of complex I (FMN and 8 -9 ironsulfur clusters) resides in the membrane domain (3, 10). The Fe 4 S 4 cluster N2 is the last component in a "wire" of seven iron-sulfur clusters connecting FMN, the immediate oxidant for NADH, and substrate ubiquinone (3,11). While all other clusters in this chain have a redox midpoint potential of around Ϫ250 mV, iron-sulfur cluster N2 has a significantly more positive E m7 of typically Ϫ150 mV (12, 13). Moreover, the redox midpoint potential of cluster N2 exhibits pronounced pH dependence between pH 6 and 8 (12). Based on this "redoxBohr" effect (14) and the comparably more positive potential, cluster N2 has been implicated as a key component of the proton pump (6, 15).The structural model for the ubiquinone reducing catalytic core of complex I (1, 16) that had been deduced from distinct homologies between [NiFe] hydrogenase and complex I (17, 18) was now confirmed by the structure of the peripheral arm of complex I (3). Like the proximal iron-sulfur cluster of hydrogenase, iron-sulfur cluster N2 is at the interface between two subunits, the PSST subunit and the 49-kDa subunit (Fig. 1). In water-soluble [NiFe] hydrogenases a conserved histidine of the l...

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“…The 18 to 20-kDa polypeptide was the only one that reacted exclusively with NADPH. In earlier publications (Belogrudov and Hatefi 1994;Belogrudov and Hatefi 1996) the investigators mentioned that a 20-kDa subunit was one of the 31 subunits of the hydrophobic-protein (HP) domain that can be obtained by resolution of Complex I with chaotropic agents (Hatefi and Stempel 1967) and that this subunit contained a motif of three Cys residues that could possibly bind a tetranuclear Fe-S cluster. Obviously, this subunit is the PSST subunit.…”

Section: Pyridine-nucleotide Binding Sites In Complex Imentioning

confidence: 99%

“…Obviously, this subunit is the PSST subunit. The iron-sulfur protein (IP) domain of Complex I also contains a 18-kDa subunit (Belogrudov and Hatefi 1994) with AQDQ as the first four residues . This subunit is proposed to modulate the activity of Complex I by way of proteinekinase-A-mediated phosphorylation of a Ser residue by cAMP in its C-terminus (Papa et al 2008).…”

Section: Pyridine-nucleotide Binding Sites In Complex Imentioning

confidence: 99%

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

Albracht

2010

J Bioenerg Biomembr

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Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The longknown reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430-1436 disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90Å long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature.

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Catalytic Sector of Complex I (NADH:Ubiquinone Oxidoreductase):Subunit Stoichiometry and Substrate-Induced Conformation Changes

Cited by 112 publications

References 38 publications

Substrate-induced Conformational Change in Bacterial Complex I

Substrate-induced Conformational Change in Bacterial Complex I

The Redox-Bohr Group Associated with Iron-Sulfur Cluster N2 of Complex I

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

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