MITOCHONDRIAL NADH-UBIQUINONE OXIDOREDUCTASE (COMPLEX-I) - EFFECTS OFSUBSTRATES ON THE FRAGMENTATION OF SUBUNITS BY TRYPSIN

It has been shown that treatment of bovine mitochondrial complex I (NA DH-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 cont ain electron carriers, such as FMN and iron-sulfur clusters, as well a s subunits that lack electron carriers. Results shown elsewhere on cha nges in the cross-linking pattern of complex I subunits when the enzym e was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1 994) Biochemistry 33, 4571-4576) also indicated that complex I undergo es extensive conformation changes when reduced by substrate. Furthermo re, we had previously shown that in submitochondrial particles the aff inity of complex I for NAD increases by greater than or equal to 20-fo ld in electron transfer from succinate to NAD when the particles are e nergized by ATP hydrolysis. Together, these results suggest that energ y coupling in complex I may involve protein conformation changes as a key step. In addition, it has been shown here that treatment of comple x I with trypsin in the presence of NADPH, but not NADH or NAD(P), pro duced from the 39-kDa subunit a 33-kDa degradation product that resist ed further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bou nd to a NADP-agarose affinity column, and could be eluted with a buffe r containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved i n intramitochondrial fatty acid synthesis.