Abstract
NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies.
Highlights
NADH-ubiquinone oxidoreductase is the largest (ϳ1 MDa) and the least characterized complex of the mitochondrial electron transport chain
Together with the other protonpumping OXPHOS-ETC complexes, ubiquinol-cytochrome c oxidoreductase and cytochrome c oxidase, complex I is responsible for building up a large proton electrochemical gradient that is harvested by ATP synthase for ATP production [1]
Progress has been made in our understanding of the mechanism of the OXPHOS-ETC complexes, including high resolution structures of mammalian mitochondrial complexes II, III, and IV (6 –9), until recently only medium resolution structures were available for intact complexes I and V (10 – 14)
Summary
NADH-ubiquinone oxidoreductase (complex I) is the largest (ϳ1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Progress has been made in our understanding of the mechanism of the OXPHOS-ETC complexes, including high resolution structures of mammalian mitochondrial complexes II, III, and IV (6 –9), until recently only medium resolution structures were available for intact complexes I and V (10 – 14). Because of its large ϳ1-MDa size, its 45 total subunits (44 unique subunits with SDAP present in two copies [11]) as well as the separation of its redox cofactors and proton pumps in large hydrophilic and hydrophobic arms, complex I remains the least well mechanistically and structurally characterized of the OXPHOS-ETC complexes. The main process by which ATP is produced involves the five large membrane protein complexes of the oxidative phosphorylation electron transport chain
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