GroEL-assisted refolding of adrenodoxin during chemical cluster insertion

Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadre nodoxin was carried out in the presence of the nonhomologous chaperone prot ein GroEL and of the cochaperone GroES, both in the presence and in the abs ence of ATP. The approach used here was different from the one characterizi ng studies on chaperone activity, as we used an adrenodoxin apoprotein, dev oid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possibl e scaffolding role for two bacterial sulfur-transferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Es cherichia coli, was also investigated in the absence of the enzyme substrat es. The extent and the rate of adrenodoxin refolding following cluster inse rtion was measured by spectroscopy and by monitoring the activity recovery in a NADPH-cytochrome c reduction assay. These measurements were carried ou t on the unresolved reaction mixture and on the adrenodoxin-containing frac tion obtained by HPLC fractionation of the reconstitution mixture at differ ent reaction times. The rate and extent of cluster insertion and activity r ecovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanes e, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical re constitution of apoadrenoxin in the presence of GroEL is therefore attribut able to a scaffolding effect of this protein.