Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc

Abstract

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.

Congenital Adrenal Hyperplasia due to 11-Hydroxylase Deficiency – Insights from Two Novel <i>CYP11B1</i> Mutations (p.M92X, p.R453Q)

“…This domain is required for recognition and interaction of Adx with its redox partner’s adrenodoxin reductase (ADR) and the cytochrome P450 monoxygenase. The recognition is mainly based on electrostatic interactions of negatively charged amino acids on the surface of Adx with positively charged amino acids of ADR or of the cytochrome P450 enzyme, respectively [20,21]. A variety of interaction sites of bovine CYP11A1 and Adx have been suggested [22,23].…”

The competition between chemistry and biology in assembling iron–sulfur derivatives. Molecular structures and electrochemistry. Part II. {[Fe2S2](SγCys)4} proteins

“…In contrast to the 1:1 complex of HAdx with cytochrome P450 scc , it is assumed that the interaction of BAdx with cytochrome P450 scc does not afford a true coordination complex but a simple reciprocal approach of the two components caused by the electrostatic interaction between the negatively charged amino acid residues of BAdx and the positively charged residues of cytochrome P450 scc [223]. Such an assumption is supported by the solution NMR structure of the system constituted by truncated 4-108 BAdx (doubly mutated as Leu80Cys/Cys95Ser) and cytochrome c (in the mutated form Val28Cys) [224].…”

Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

“…At the electron transfer site (III), the hydrogen bond between E47 of Adx and N63 of AdR [43] can also be formed to link E557 of Etp1 fd with N63 of AdR. N-terminally, the vicinity of the [2Fe-2S]…”