Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation.
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.
From the Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria.
James A
JA
Letts
the Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom, and.
Gianluca
G
Degliesposti
From the Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria,; the Medical Research Council Mitochondrial Biology Unit, Cambridge CB2 0XY, United Kingdom.
Karol
K
Fiedorczuk
the Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom, and.
Mark
M
Skehel
From the Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria,. Electronic address: sazanov@ist.ac.at.
Leonid A
LA
Sazanov
D000818:Animals; D002417:Cattle; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D008929:Mitochondria, Heart / Q000201:enzymology*; D012756:Sheep
D042967:Electron Transport Complex I
complex I; electron transfer; membrane protein; mitochondrial respiratory chain complex; proton pump; respiratory chain
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