Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc - Mass spectrometry and Edman degradation ofcomplexes of the steroidogenic hydroxylase system

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cyto chromes P450 constitute the mitochondrial steroid hydroxylase system of ver tebrates. During the reaction cycle, adrenodoxin transfers electrons from t he FAD of adrenodoxin reductase to the heme iron of the catalytically activ e cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organize d cluster model of all three components has been discussed to explain elect ron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminoprop yl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectro metry and X-ray crystallography a crosslink between adrenodoxin reductase L ys27 and adrenodoxin Asp39 was detected, establishing a secondary polar int eraction site between both molecules. No crosslink exists in the primary po lar interaction site around the acidic residues Asp76 to Asp79 of adrenodox in. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hyd roxylase activity could be detected in an adrenodoxin -P450scc complex/adre nodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc comple x prevents electron transfer by a putative shuttle mechanism. Thus, chemica l crosslinking provides evidence favoring the shuttle model over the cluste r model for the steroid hydroxylase system.