ISOALLOXAZINE RING OF FAD IS REQUIRED FOR THE FORMATION OF THE CORE IN THE HSP60-ASSISTED FOLDING OF MEDIUM-CHAIN ACYL-COA DEHYDROGENASE SUBUNIT INTO THE ASSEMBLY COMPETENT CONFORMATION IN MITOCHONDRIA

We studied the role of FAD in the intramitochondrial folding and assem bly of medium chain acyl-CoA dehydro genase (MCAD), a homotetrameric m itochondrial enzyme containing a molecule of non-covalently bound FAD/ monomer. In the MCAD molecule, FAD is buried in a crevice containing t he active center. We have previously shown that upon import into mitoc hondria, newly processed MCAD is first incorporated into a high molecu lar weight (hMr) complex and that the hMr complex mainly consisted of MCAD heat shock protein 60 (hsp60) complex (Saijo, T., Welch, W. J., a nd Tanaka, K (1994) J. Biol. Chem. 269, 4401-4408). In the present stu dy, we incubated in vitro synthesized precursor MCAD with mitochondria isolated fi om normal and riboflavin-deficient rat liver for 10-60 mi n and fractionated the solubilized mitochondria using gel filtration. The amount of MCAD in the hMr complex was larger and that of tetramer was smaller in riboflavin-deficient mitochondria than in control at an y time point, In addition, riboflavin-deficient mitochondria were solu bilized after 10-min import in a buffer containing ATP and were chased in the presence of FAD, FMN, or NAD(+) or without any addition. The m itochondrial proteins were analyzed using gel filtration or immunoprec ipitated with anti-hsp60 antibody, After 60-min chase in the presence of FAD, the majority of MCAD in the complex with hsp60 was transferred to tetramer, whereas no such transfer occurred after the chase in the absence of FAD. When chase was done in the presence of FMN, a signifi cant amount of MCAD was transferred from the complex with hsp60 to tet ramer, but the transfer was not as efficient as in the presence of FAD , The chase in the presence of NAD(+) resulted in no transfer, These d ata suggest that isoalloxazine ring of FAD plays a critical role, exer ting nucleating effect, in the hsp60-assisted folding of MCAD subunit into an assembly competent conformation, probably assisting the format ion of the core.