Mitochondrial NADH-ubiquinone oxidoreductase (Complex I). Effect of substrates on the fragmentation of subunits by trypsin.
It has been shown that treatment of bovine mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576) also indicated that complex I undergoes extensive conformation changes when reduced by substrate. Furthermore, we had previously shown that in submitochondrial particles the affinity of complex I for NAD increases by >/=20-fold in electron transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve protein conformation changes as a key step. In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.
Yamaguchi
M
M
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Belogrudov
G I
GI
Hatefi
Y
Y
D000818:Animals; D002417:Cattle; D008929:Mitochondria, Heart / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D009243:NAD; D009249:NADP; D016660:NAD(P)H Dehydrogenase (Quinone); D014357:Trypsin