Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.
NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.
Finel
M
M
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Skehel
J M
JM
Albracht
S P
SP
Fearnley
I M
IM
Walker
J E
JE
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction
D005486:Flavin Mononucleotide; D016660:NAD(P)H Dehydrogenase (Quinone)