The steroid hydroxylating system of adrenal cortex mitochondria consists of the membrane-attached NADPH-dependent adrenodoxin reductase (AR), the soluble one-electron transport protein adrenodoxin (Adx), and a membrane-integrated cytochrome P450 of the CYP11 family. In the 2.3-Å resolution crystal structure of the Adx⅐AR complex, 580 In mitochondria of the adrenal cortex, the cytochrome P450 enzymes of the CYP11 family catalyze the side chain cleavage of cholesterol to form pregnenolone (P450scc, 1 CYP11A1) and are involved in the formation of cortisol (P45011␤, CYP11B1) and aldosterone (P450aldo, CYP11B2) (1). The enzymatic activity of the cytochrome P450-dependent steroid hydroxylases is based on their ability to activate molecular oxygen by reductive splitting of dioxygen. This multistep reaction requires the transfer of electrons from the flavoprotein adrenodoxin reductase (AR) via adrenodoxin (Adx) to the terminal cytochromes P450 as electron acceptors in dependence on the specific hydroxylation substrate (1-3). Several models for electron transfer have been discussed, including a shuttle model in which Adx forms consecutive 1:1 complexes (4) with AR and cytochrome P450scc and models requiring the formation of an organized 1:1:1 ternary complex (5) or a 1:2:1 quaternary complex (6) between AR, Adx, and cytochrome P450scc. Common to these models is a complex between AR and Adx during the first steps of electron transfer from the reductase to the cytochrome P450.Recently, the crystal structures of two forms of bovine adrenodoxin (7, 8) and of adrenodoxin reductase (9) were determined. These structures revealed the general topology of the two proteins and the molecular environments of the [2Fe-2S] cluster of Adx and the FAD moiety of AR. Here, we report the 2.3-Å resolution crystal structure of a cross-linked 1:1 complex of full-length Adx and AR. This structure shows the geometry of an electron transfer complex of soluble, freely dissociable proteins from a higher eukaryote for the first time, highlights structural adaptations that accompany the binding of AR to Adx, and permits us to predict electron transport paths in their complex. EXPERIMENTAL PROCEDURESSample Preparation-Recombinant bovine Adx and AR were purified and crystallized as described (10). The synthesized Adx differs from the wild-type protein by the exchange of Ser 1 for glycine and is composed of 128 amino acids, including the N-and C-terminal residues missing in the truncated adrenodoxin, Adx-(4 -108), studied earlier (7). Crosslinking of AR to Adx has also been described (11,12). The native complex is formed at low ionic strength between the two proteins, and the cross-linking was carried out with the water-soluble coupling reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, purchased from Sigma). The mixture of Adx (1.2 mol) and AR (300 nmol) was dialyzed for 18 -20 h against 20 mM potassium phosphate, pH 7.2, followed by addition of an equal volume of fresh 8 mM EDC solution in distilled water and incubation at 4°C in the dark wi...

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NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) 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 spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin 2P450scc complex/adrenodoxin 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 adrenodoxin2P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) 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 spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin 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 complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.