STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF BOVINE ADRENODOXIN REDUCTASE BY LIMITED PROTEOLYSIS

Previously, we have proposed that bovine adrenocortical mitochondrial adrenodoxin reductase may possess a domain structure, based upon the g eneration of two major peptide fragments from limited tryptic proteoly sis. In the present study, kinetic characterization of the NADPH-depen dent ferricyanide reductase activity of the partially proteolyzed enzy me demonstrates that K-m(NADPH) increases (from 1.2 mu M to 2.7 mu M), whereas V-max remains unaltered at 2100 min(-1). The two proteolytic fragments have been purified to homogeneity by reverse-phase HPLC, and amino-acid sequence analysis unambiguously demonstrates that the 30.6 kDa fragment corresponds to the amino terminal portion of the intact protein, whereas the 22.8 kDa fragment is derived from the carboxyl te rminus of the reductase. Trypsin cleavage occurs at either Arg-264 or Arg-265. Covalent crosslinking experiments using a water-soluble carbo diimide show that adrenodoxin crosslinks exclusively to the 30.6 kDa f ragment, thus implicating the N-terminal region of adrenodoxin reducta se in binding to the iron-sulfur protein. Our inability to detect cova lent carbohydrate on either intact or proteolyzed adrenodoxin reductas e prompted a re-examination of the previously reported requirement of an oligosaccharide moiety for efficient electron transfer from the red uctase to adrenodoxin. Treatment of adrenodoxin reductase with a highl y purified preparation of neuraminidase demonstrates that neither the adrenodoxin-independent ferricyanide reductase activity nor the adreno doxin-dependent cytochrome c reductase activity of the enzyme is affec ted by neuraminidase treatment.