Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

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.

Section: Discussionmentioning

confidence: 53%

“…Evidence indicating conformational changes in complex I came from studies with the bovine enzyme, where the patterns of cross-linking between subunits (23) and the extent of proteolysis (26) changed significantly in the presence of NAD(P)H, but not NAD ϩ . These data indicate that NAD(P)H binding induced significant conformational change in bovine complex I.…”

mentioning

confidence: 99%

Substrate-induced Conformational Change in Bacterial Complex I

Mamedova

Holt

Carroll

et al. 2004

“…It should be noted that structural changes seem to take place around this subunit of the enzyme complex in situ. Studies on cross-linking and susceptibility to trypsin digestion of mitochondrial complex I have shown that extensive conformational changes undergo upon reduction by substrate NADH or NADPH (51,52). These physicochemical features of the higher spin system and associated technical difficulties may partly explain why cluster N5 is only partially or hardly detectable in complex I/NDH-1 from different species.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Cluster N5 as a Fast-relaxing [4Fe-4S] Cluster in the Nqo3 Subunit of the Proton-translocating NADH-ubiquinone Oxidoreductase from Paracoccus denitrificans

Yano

Nakamaru‐Ogiso

et al. 2003

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, The energy-transducing NADH-quinone oxidoreductase is located at an entry point of the respiratory chain of mitochondria and bacteria, and catalyzes the redox coupled H ϩ or Na ϩ ion transport reaction (1). The enzyme complex is one of the largest and most complicated membrane-bound respiratory chain enzymes ever known. Mitochondrial enzyme (complex I) is composed of at least 46 subunits (2, 3), whereas the bacterial counterpart (NDH-1) such as those from Paracoccus denitrificans and Thermus thermophilus consists of only 14 subunits (4). All 14 subunits of the NDH-1 are homologous to their mitochondrial counterparts. Complex I/NDH-1 share many structural and enzymatic properties. Complex I/NDH-1 is composed of two distinct domains, a hydrophilic extramembrane domain (promontory part) and a hydrophobic membrane domain. Each domain plays a distinct role in the energy transducing reaction (5-9). Generally, the enzyme complexes contain the same type and number of redox components: one non-covalently bound FMN and up to eight iron-sulfur clusters (10). All of these redox cofactors are located in the promontory part where the oxidation of NADH and the subsequent electron transfer reaction takes place toward the membrane part. In the membrane part, a lipid-soluble quinone molecule accepts electrons. In this terminal reaction step, some membrane-bound quinone species are suggested to be involved (11, 12); however, these quinone-binding sites remain to be located. These redox reactions are endergonic, releasing free energy that is used to transport ions (H ϩ or Na ϩ ) through channels, which are thought to traverse the membrane domain. In the case of bovine heart mitochondrial complex I, ϳ4 H ϩ are transported during the electron transfer from NADH to quinone pool. However, the energy coupling mechanism has not been solved experimentally. The understanding of this process is a crucial issue of complex I bioenergetics.Among the eight iron-sulfur clusters common in complex I/NDH-1 family, six clusters are generally detectable by EPR spectroscopy in situ, namely two [2Fe-2S] clusters, N1a and N1b, and four [4Fe-4S] clusters, N2, N3, N4, and N5 (10). Thus far, subunit locations of clusters N1a, N1b, N3, and N4 have tentatively been assigned based upon biochemical/physicochemical investigations of mammalian and microorganisms (10) and a series of expression studies of the individual putative iron-sulfur cluster-binding subunits of the P. denitrificans enzyme (13-15). Furthermore, two additional [4Fe-4S] clusters, which have not been detected by EPR in situ, are coordinated in the TYKY/Nqo9/NuoI subunit (nomenclature used for bovine heart mi...

“…Complex I catalyzes the transfer of electrons from matrix NADH to membrane ubiquinone coupled to the translocation of four protons across the membrane (10,11). Numerous hypothetical schemes for the coupling mechanism of complex I can be found in the literature; the most recent ones involve long range conformational changes in the enzyme complex rather than variations of a classical redox loop or pump (12)(13)(14)(15). In the presence of ⌬H ϩ across the membrane, the enzyme is also able to catalyze the reverse reaction and reduce NAD ϩ by the quinol pool (16,17).…”

mentioning

confidence: 99%

Superoxide Radical Formation by Pure Complex I (NADH:Ubiquinone Oxidoreductase) from Yarrowia lipolytica

Galkin¹,

Brandt

2005

149

110

Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I (NADH:ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex I from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH 2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.Over the last decade the processes leading to the production of superoxide and other reactive oxygen species (ROS) 1 have gained much attention. ROS seem to be involved in apoptosis, the development of various pathological states, aging, and the regulation of cell metabolism. It is generally accepted that production of reactive oxygen species is an inherent property of the mitochondrial respiratory chain of eucaryotic cells. Oxidation of certain redox centers in complex I and III by molecular oxygen results in the production of superoxide anion radical O 2. (see Ref. 1 for a review). Superoxide can then convert into hydrogen peroxide, the highly active hydroxyl radical (OH ⅐ ), and other ROS. It has been shown that O 2 . production by complex I occurs in the mitochondrial matrix, whereas the cytochrome bc 1 complex reduces oxygen primarily on the intermembrane side (2, 3) (see, however, Ref. 4). It has been demonstrated for the cytochrome bc 1 complex that oxygen reduction occurs at the Q P site and is increased markedly under conditions of "oxidant-induced reduction" (5, 6). However, much less is known about the site and mechanism of O 2 . generation in complex I. Thermodynamically, any of the complex I redox centers in the reduced state is capable of donating an electron to molecular oxygen to form a superoxide anion. Eucaryotic NADH:ubiquinone oxidoreductase (complex I or type I NADH dehydrogenase) is the largest and most complex enzyme of the respiratory chain, residing in the inner membrane of mitochondria. In mammals, the enzyme is composed of 46 different subunits (7) and contains non-covalently bound FMN and up to eight iron-sulfur clusters as redox cofactors. Two complex I-associated, electron paramagnetic resonancedetectable semiquinone species with different spin relaxation times have been characterized (8, 9). Complex I catalyzes the transfer of electrons from matrix NADH to membrane ubiquinone coupled to the translocation of four protons across the membrane (10, 11...