We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.