The Loop Region Covering the Iron-Sulfur Cluster in Bovine Adrenodoxin Comprises a New Interaction Site for Redox Partners

Abstract

The amino acid in position 49 in bovine adrenodoxin is conserved among vertebrate [2Fe-2S] ferredoxins as hydroxyl function. A corresponding residue is missing in the cluster-coordinating loop of plant-type [2Fe-2S] ferredoxins. To probe the function of Thr-49 in a vertebrate ferredoxin, replacement mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49⌬, were generated and expressed in Escherichia coli. CD spectra of purified proteins indicate changes of the [2Fe-2S] center geometry only for mutant T49⌬, whereas NMR studies reveal no transduction of structural changes to the interaction domain. The redox potential of T49⌬ (؊370 mV) is lowered by ϳ100 mV compared with wild type adrenodoxin and reaches the potential range of planttype ferredoxins (؊305 to ؊455 mV). Substitution mutants show moderate changes in the binding affinity to the redox partners. In contrast, the binding affinity of T49⌬ to adrenodoxin reductase and cytochrome P-450 11A1 (CYP11A1) is dramatically reduced. These results led to the conclusion that Thr-49 modulates the redox potential in adrenodoxin and that the cluster-binding loop around Thr-49 represents a new interaction region with the redox partners adrenodoxin reductase and CYP11A1. In addition, variations of the apparent rate constants of all mutants for CYP11A1 reduction indicate the participation of residue 49 in the electron transfer pathway between adrenodoxin and CYP11A1.[2Fe-2S] ferredoxins are found in all organisms from archaea and bacteria to higher plants and animals and function as mediators of electron transfer in a range of multicomponent redox systems (1). The redox active prosthetic group in this class of ferredoxins is characterized by an iron-sulfur cluster, consisting of two non-heme iron ions ligated to thiolate side chains of four cysteines of the polypeptide, bridged by two inorganic sulfide ions.[2Fe-2S] ferredoxins are classified by structure and function into plant-type ferredoxins and vertebrate-type ferredoxins. Vertebrate-type [2Fe-2S] ferredoxins, present in oxygenase systems of bacteria and vertebrates, transfer electrons from a NAD(P)H-dependent ferredoxin reductase to different cytochrome P450 enzymes (2). In vertebrates, ferredoxins of the [2Fe-2S] protein family are present in adrenal cortex, placenta, liver, kidney, and brain (3), where they participate in cytochrome P450-catalyzed hydroxylation reactions to produce steroid hormones, vitamin D metabolites, and bile acids.Bovine Adx 1 as a member of vertebrate [2Fe-2S] ferredoxins, functions as electron mediator from the isoalloxazin system of adrenodoxin reductase (AdR) to the heme iron of two cytochromes P450, CYP11A1 and CYP11B1, localized in the inner mitochondrial membrane of the adrenal cortex (4). CYP11A1 converts cholesterol to pregnenolone, and CYP11B1 catalyzes the 11␤-hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol and the production of aldosterone.The crystal structures of a truncated bovine Adx (5) and of a full-length Adx (6) determined at 1.85-and 2.5-Å resolution, r...

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

“…This complex is formed after cytochrome reduction. 27,36 Binding of CO to the reduced heme iron of CYP11A1 can be considered irreversible. Previous stopped-flow experiments 38,39,40 CYP11A1 by Adx.…”

“…27,30 Amino acid substitutions were generated with the QuikChange site directed mutagenesis kit (Stratagene; La Jolla, CA). Oligonucleotides containing the appropriate mutations were synthesized by BioTez GmbH (BioTez; Berlin, Germany).…”

“…This kinase is a ubiquitous Ser and Thr phosphorylating enzyme that possesses a very simple phosphorylation motif ([S/T]-X-X-[D/E]). 26,27 Since the presence of CK2 in the mitochondria has been demonstrated, 28 phosphorylation of components of the mitochondrial steroid hydroxylating ET chain by CK2 could influence the ET. In fact, it was shown that CK2 phosphorylated Adx and a mutant that simulates phosphorylation at residue 71, Adx-T71E, both led to an enhanced formation of pregnenolone from cholesterol as measured in an in vitro reconstituted CYP11A1 system (approximately 1.5-fold).…”

“…21,22 Additional sites involved in redox partner binding on the core domain have been identified in the crystal structure of a crosslinked 1:1 complex of Adx and AdR 23 and by site-directed mutagenesis studies. 24,27 These newly discovered interaction sites comprise a second acidic patch around position Asp-39, the loop surrounding the ironsulfur cluster, 24,27 and involve C-terminal residues of the ferredoxin. 25 Recent investigations 1 have shown that Adx is phosphorylated in vitro at position T71 by casein kinase 2 (CK2).…”

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

“…Recombinant Adx, Etp1 fd and Arh1 were purified as described [7,21,22]. The protein concentrations were calculated with ε 414 = 9.8 (mM cm) -1 for Etp1 fd [21] and Adx with ε 450 = 11.3 (mM cm) -1 for Arh1 [7].…”