Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

Abstract

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...

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

“…To understand the mode of protein-protein interactions in the AdR-Adx-CYP11A1 system, we compared the CYP11A1-Adx complex with the AdR-Adx complex (37). Adx residues involved in the interaction interface with AdR and CYP11A1 greatly overlap (Fig.…”

The ferredoxin reductase gene is regulated by the p53 family and sensitizes cells to oxidative stress-induced apoptosis

“…FR, a 50-kD mitochondrial flavoprotein, participates in the cleavage of cholesterol side chain to form pregnenolone, cortisol, and aldosterone in a multi-step biosynthesis, which generates molecular oxygen by reductive splitting of dioxygen and requires the transfer of electrons by FR via ferredoxin to cytochrome P450 (Muller et al, 2001). Here, we and Hwang et al (2001) have found that in addition to the biosynthesis of steroid hormones, FR sensitizes cancer cells to ROSinduced apoptosis.…”

Protein Dynamics Enhance Electronic Coupling in Electron Transfer Complexes

“…An in-depth understanding of these reactions requires knowledge of the role of protein dynamics in complex assembly and the geometries of the complex that are compatible with interprotein electron transfer (eT). 1 A role for interdomain flexibility has been demonstrated in eT both within a single molecule (3)(4)(5) and between molecules (6).…”