Mitochondrial Steroid Hydroxylase Systems Research Articles

Self-association of adrenodoxin studied by using analytical ultracentrifugation

The mitochondrial steroid hydroxylase system of vertebrates utilizes adrenodoxin (Adx), a small iron–sulfur cluster protein of about 14 kDa as an electron carrier between a reductase and cytochrome P450. Although the crystal structure of this protein has been elucidated, the solution structure of Adx was discussed contrary in the literature [I.A. Pikuleva, K. Tesh, M.R. Waterman, Y. Kim, The tertiary structure of full-length bovine adrenodoxin suggests functional dimers, Arch. Biochem. Biophys. 373 (2000) 44–55; D. Beilke, R. Weiss, F. Löhr, P. Pristovsek, F. Hannemann, R. Bernhardt, H. Rüterjans, A new electron mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin, Biochemistry 41 (2002) 7969–7978]. Therefore, it was necessary to study the self-association of this protein by using analytical ultracentrifugation over a larger concentration range. As could be demonstrated in sedimentation velocity experiments, as well as sedimentation equilibrium runs with explicit consideration of thermodynamic non-ideality, the full-length protein (residues 1–128) in the oxidized state resulted in a monomer–dimer equilibrium ( K a ~ 3 × 10 2 M − 1 ). For truncated Adx (1–108), as well as the reduced Adx, the association behavior was strongly reduced. The consequences of this behavior are discussed with respect to the physiological meaning for the Adx system.

Light-Induced Reduction of Bovine Adrenodoxin via the Covalently Bound Ruthenium(II) Bipyridyl Complex: Intramolecular Electron Transfer and Crystal Structure

Bovine adrenodoxin (Adx) plays an important role in the electron-transfer process in the mitochondrial steroid hydroxylase system of the bovine adrenal cortex. Using electron paramagnetic resonance (EPR) spectroscopy, we showed that photoreduction of the [2Fe-2S] cluster of Adx via (4'-methyl-2,2'-bipyridine)bis(2,2'-bipyridine)ruthenium(II) [Ru(bpy)2(mbpy)] covalently attached to the protein surface can be used as a new approach to probe the "shuttle" hypothesis for the electron transfer by Adx. The 1.5 A resolution crystal structure of a 1:1 Ru(bpy)2(mbpy)-Adx(1-108) complex reveals the site of modification, Cys95, and allows to predict the possible intramolecular electron-transfer pathways within the complex. Photoreduction of uncoupled Adx, mutant Adx(1-108), and Ru(bpy)2(mbpy)-Adx(1-108) using safranin T as the mediating electron donor suggests that two electrons are transferred from the dye to Adx. The intramolecular photoreduction rate constant for the ruthenated Adx has been determined and is discussed according to the predicted pathways.

Inhibition of aldosterone biosynthesis by staurosporine

Staurosporine (STS) is a very potent broad-range kinase inhibitor, and its antiproliferative properties made it a lead compound for protein kinase C (PKC) inhibitors with therapeutic potential. Because STS also causes hypotension, we investigated in this study whether it directly interferes with the terminal steps of aldosterone biosynthesis; these are catalysed by a mitochondrial steroid hydroxylase system consisting of adrenodoxin reductase, adrenodoxin, and the cytochrome P450 enzyme hCYP11B2 (aldosterone synthase). Here we demonstrate that nanomolar concentrations of STS significantly reduced aldosterone synthase activity in transiently transfected COS-1 cells and in stably transfected V79MZh11B2 cells (IC50 = 11 nM). However, STS did not inhibit bovine aldosterone synthase in a reconstituted steroid hydroxylation assay. In transiently transfected COS-1 cells, the protein level of adrenodoxin (but not that of adrenodoxin reductase or of hCYP11B2) was significantly reduced after treatment with 2 nM STS. Finally, we show that STS treatment (1 microg/day) of mice reduced their aldosterone/renin ratio by almost 50% (p = 0.015). To the best of our knowledge, this is the first report of a direct in vivo effect of STS on the renin-angiotensin-aldosterone system. We conclude (i) that the hypotensive effect of staurosporine is at least partly due to inhibition of aldosterone biosynthesis via adrenodoxin depletion, and (ii) that aldosterone biosynthesis can be regulated in vivo at the level of adrenodoxin availability.

Modeling of electrostatic recognition processes in the mammalian mitochondrial steroid hydroxylase system

Adrenodoxin reductase (AR) and adrenodoxin (Adx) are components of the mammalian mitochondrial steroid-hydroxylating system. Crystal structures of Adx, AR and a cross-linked Adx–AR complex have recently been determined. Based on these, we have carried out a modeling and docking study to characterize the recognition between AR, Adx and cytochrome c (Cytc). To rationalize the recognition process, electrostatic potentials were calculated by solving the Poisson–Boltzmann equations. In the Adx–AR complex modeled, a negatively charged surface of Adx recognizes a positive surface of AR, as in the crystal structure of the Adx–AR complex, proving the correct parameterization for the energy calculations. After forming salt bridges between the polar primary binding sites of Adx and AR, charge compensation causes a domain movement in AR, which closes the binding cleft by 2–4 Å. Thereby, a secondary polar binding site is closed and the electron transfer pathways between the FAD of AR and the [2Fe–2S] cluster of Adx are adjusted. Next, the model structure of a complex between Adx and Cytc was derived. The lowest-energy complex between Adx and Cytc matches earlier chemical modification and cross-linking experiments, which proposed polar interactions of Lys13, Lys27, Lys72 and Lys79 of Cytc with acidic residues in Adx. Because of the short distance of 9.4 Å between the redox centers, a complex, productive in electron transfer via a different outlet pathway from the inlet route in Adx, is expected. However, a ternary complex cannot be formed between the Adx–AR complex and Cytc because of steric hindrance. Therefore, a shuttle model for the role of Adx in the electron transfer process to Cytc is preferable to a relay model. In addition, no preferable docking site could be detected for a second Adx when probing the Adx–AR complex, which is required for a quaternary organized-cluster model of all redox partners of the hydroxylase system.

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

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.

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc Mass spectrometry and Edman degradation of complexes of the steroidogenic 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.

Evidence for the Cluster Model of Mitochondrial Steroid Hydroxylase System Derived from Dissociation Constants of the Complex between Adrenodoxin Reductase and Adrenodoxin

Using biotinylated adrenodoxin and avidin–Sepharose 4B, dissociation constants for the complex between adrenodoxin reductase and adrenodoxin in the oxidized and reduced states were determined as 50 ± 11 and 296 ± 44 nM, respectively. Concentrations of adrenodoxin reductase/adrenodoxin in the matrix fraction from bovine adrenal cortex, liver, and kidney mitochondria were determined to be 20.2 ± 10.6 μM/120 ± 23 μM, 0.17 ± 0.06 μM/1.79 ± 0.24 μM, and 0.40 ± 0.23 μM/1.33 ± 0.26 μM, respectively. The calculation of the percentage of adrenodoxin reductase in the complex form in the reduced state showed that it is higher than 99% in adrenal cortex mitochondria, providing clear evidence for the cluster model for the mitochondrial steroid hydroxylase system.

Molecular recognition and electron transfer in mitochondrial steroid hydroxylase systems

Mitochondrial monooxygenase systems are involved in the biosynthesis of glucocorticoids, mineralocorticoids, bile acids, and 1,25-dihydroxyvitamin D. The reactions are catalyzed by specific P450 enzymes that receive reducing equivalents via NADPH-ferredoxin oxidoreductase (adrenodoxin reductase) and ferredoxin (adrenodoxin). Although the three-dimensional structures of the individual components have not yet been solved, methods of expressing recombinant forms of these enzymes in Escherichia coli have allowed the use of site-directed mutagenesis to investigate the roles of specific amino acids in protein binding interactions, electron transfer, and catalysis. These studies have identified key charged residues in NADPH-ferredoxin oxidoreductase, ferredoxin, and P450scc, which are involved in electrostatic interactions critical for recognition, high-affinity binding, and electron transfer. The finding that the binding sites on ferredoxin for NADPH-ferredoxin oxidoreductase and P450 show significant overlap supports the proposed function for ferredoxin as a mobile electron shuttle between the reductase and P450 enzymes and is consistent with ferredoxin's role in serving multiple P450 isoforms.

Existence of adrenodoxin‐like peptide in teleost fishes: Immunohistochemical evidence

AbstractThis communication reports the first evidence, based on immunohistochemistry, for the presence of adrenodoxin‐like protein in poikilothermic vertebrates, two species of teleost fishes. Adrenodoxin (adrenal ferredoxin) is an iron‐sulphur protein present in the mitochondrial matrix of mammalian and avian adrenal cortex. Presence of this protein in the adrenal cortex or its equivalent (interrenal) of poikilothermic vertebrates is unknown to date. Contrary to the expectation, an earlier study using EPR spectrometry failed to demonstrate adrenodoxin in tiger shark and green turtle leading to the hypothesis that mitochondrial steroid hydroxylase systems (Type II enzymes) in poikilotherms might be similar to the microsomal counterparts (Type I enzymes) which does not involve adrenodoxin. ABC method of immunohistochemistry in the present study demonstrated that antiserum to bovine adrenodoxin reacts specifically and intensely with interrenal cells of the Asian seabass (Lates calcarifer) and the tilapia (Oreochromis mossambicus), indicating that adrenodoxin or its homologue is present in the poikilotherms also. The results further suggest the existence of a distinct electron transport system for mitochondrial steroid hydroxylation in poikilotherms as in mammals. © Wiley‐Liss, Inc.

Cell-free synthesis of precursor forms of mitochondrial steroid hydroxylase enzymes of the bovine adrenal cortex

Newly synthesized, [ 35S]methionine-labeled cholesterol side-chain cleavage cytochrome P-450, 11β-hydroxylase cytochrome P-450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P-450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [ 35S]methionine-labeled 11β-hydroxylase cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments.

Topological studies of the steroid hydroxylase complexes in bovine adrenocortical mitochondria.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria was studied by using nonpenetrating artificial electron acceptors and the impermeable protein reagent diazobenzenesulfonate. Inhibition of steroid hydroxylase activity by ferricyanide and dichlorophenolindophenol sulfonate was only observed in mitochondria which had been damaged by various techniques. Intact mitochondria were not inhibited by these reagents. The reaction was monitored by oxygen uptake due to hydroxylation of deoxycorticosterone, as well as P-450 reduction and corticosterone formation. The results obtained were similar regardless of how the activity was measured. Labeling of the mitochondria with the nonpenetrating protein reagent diazobenzenesulfonate also inhibited P-450 reduction and corticosterone formation in mitochondria which had been damaged prior to addition of this reagent. Intact mitochondria which were labeled with this reagent showed very little inhibition of both activities. These results strongly suggest that all protein components of the steroid 11beta-hydroxylase system are located on the matrix side of the mitochondrial inner membrane. The inability of ferricyanide, dichlorophenolindophenol sulfonate, and diazobenzenesulfonate to inhibit the malate-dependent reduction of P-450 in intact mitochondria implies that all the P-450-dependent mitochondrial steroid hydroxylase systems are located on the matrix side of the inner mitochondrial membrane.