Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding.

The improper maintenance of the bioactivated form of vitamin-D (1α,25(OH)2D) may result in vitamin-D insufficiency and therefore compromise the absorption of dietary calcium. A significant regulator of vitamin-D metabolism is the inactivating function of the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1). In humans, CYP24A1 carries out hydroxylation of carbon-23 (C23) or carbon-24 (C24) of the 1α,25(OH)2D side chain, eventually resulting in production of either an antagonist of the vitamin-D receptor (C23 pathway) or calcitroic acid (C24 pathway). Despite its importance to human health, the human isoform (hCYP24A1) remains largely uncharacterized due in part to the difficulty in producing the enzyme using recombinant means. In this study, we utilize a cleavable fusion with the cognate redox partner, human Adx (hAdx), to stabilize hCYP24A1 during production. The subsequent cleavage and isolation of active hCYP24A1 allowed for an investigation of substrate and analog binding, enzymatic activity, and redox partner recognition. We demonstrate involvement of a nonpolar contact involving Leu-80 of hAdx and a nonconserved proximal surface of hCYP24A1. Interestingly, shortening the length of this residue (L80V) results in enhanced binding between the CYP-Adx complex and 1α,25(OH)2D yet unexpectedly results in decreased catalysis. The same mutation has a negligible effect on rat CYP24A1 (a C24-hydroxylase), indicating the presence of a species-specific requirement that may correlate with differences in regioselectivity of the reaction. Taken together, this work presents an example of production of a challenging human CYP as well as providing details regarding hydrophobic modulation of a CYP-Adx complex that is critical to human vitamin-D metabolism.

Structural and functional insights into aldosterone synthase interaction with its redox partner protein adrenodoxin.

Aldosterone is the major mineralocorticoid in the human body controlling blood pressure and salt homeostasis. Overproduction of aldosterone leads to primary aldosteronism, which is the most common form of secondary hypertension with limited treatment options. Production of aldosterone by cytochrome P450 11B2 (CYP11B2, aldosterone synthase) requires two reduction events with the electrons delivered by the iron/sulfur protein adrenodoxin. Very limited information is available about the structural and functional basis of adrenodoxin/CYP11B2 interaction, which impedes the development of new treatment options for primary aldosteronism. A systematic study was carried out to determine if adrenodoxin interaction with CYP11B2 might also have an allosteric component in addition to electron transfer. Indeed, local increases in adrenodoxin concentration promote binding of the substrate 11-deoxycorticosterone and the inhibitor osilodrostat (LCI699) in the active site-over 17 Å away-as well as enhance the inhibitory effect of this latter drug. The CYP11B2 structure in complex with adrenodoxin identified specific residues at the protein-protein interface interacting via five salt bridges and four hydrogen bonds. Comparisons with cholesterol-metabolizing CYP11A1 and cortisol-producing CYP11B1, which also bind adrenodoxin, revealed substantial structural differences in these regions. The structural and functional differences between different P450 interactions with adrenodoxin may provide valuable clues for an orthogonal treatment approach for primary aldosteronism by specifically targeting the interaction between CYP11B2 and adrenodoxin.

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species.

Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH)2D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH)2D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.

Iron-sulfur cluster biogenesis and trafficking in mitochondria.

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.

Active Site Structures of CYP11A1 in the Presence of Its Physiological Substrates and Alterations upon Binding of Adrenodoxin.

The rate-limiting step in the steroid synthesis pathway is catalyzed by CYP11A1 through three sequential reactions. The first two steps involve hydroxylations at positions 22 and 20, generating 20(R),22(R)-dihydroxycholesterol (20R,22R-DiOHCH), with the third stage leading to a C20-C22 bond cleavage, forming pregnenolone. This work provides detailed information about the active site structure of CYP11A1 in the resting state and substrate-bound ferric forms as well as the CO-ligated adducts. In addition, high-quality resonance Raman spectra are reported for the dioxygen complexes, providing new insight into the status of Fe-O-O fragments encountered during the enzymatic cycle. Results show that the three natural substrates of CYP11A1 have quite different effects on the active site structure, including variations of spin state populations, reorientations of heme peripheral groups, and, most importantly, substrate-mediated distortions of Fe-CO and Fe-O2 fragments, as revealed by telltale shifts of the observed vibrational modes. Specifically, the vibrational mode patterns observed for the Fe-O-O fragments with the first and third substrates are consistent with H-bonding interactions with the terminal oxygen, a structural feature that tends to promote O-O bond cleavage to form the Compound I intermediate. Furthermore, such spectral data are acquired for complexes with the natural redox partner, adrenodoxin (Adx), revealing protein-protein-induced active site structural perturbations. While this work shows that Adx has an only weak effect on ferric and ferrous CO states, it has a relatively stronger impact on the Fe-O-O fragments of the functionally relevant oxy complexes.

Molecular Recognition in Mitochondrial Cytochromes P450 That Catalyze the Terminal Steps of Corticosteroid Biosynthesis.

The mitochondrial cytochromes P450 11B1 and P450 11B2 are responsible for the final stages of cortisol and aldosterone synthesis, respectively. Dysregulation of both enzymes has been implicated in secondary forms of hypertension. Molecular recognition of the cytochromes P450 with their corresponding redox partner is a key step in the catalytic cycle, yet the precise nature of the interaction of P450 11B1 or P450 11B2 with their proximal partner, adrenodoxin (Adx), is still unknown. Here, we obtained P450 11B1·Adx2 and P450 11B2·Adx2 complexes using the zero-length cross-linker ethyl-3-[3-(dimethylamino)propyl]carbodiimide, which formed best under low-ionic strength conditions. R-to-K mutations were introduced into the P450s at residues predicted to form salt bridges with Adx and allow cross-linking with the carbodiimide reagent. Mass spectrometric analysis of the chymotrypsin-digested ternary complexes identified seven cross-linked peptide pairs. Consistent with the electrostatic interaction of K370 in P450 11B1-WT and K366 in P450 11B2-R366K with D79 of Adx, Adx mutation L80K abolished complex formation. Using these sites of interaction as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the P450 11B1·Adx2 complex. The appositional surfaces include R/K366, K370, and K357 of P450 11B1, which interact with D79, D76, and D113 (second molecule) of Adx, respectively. Similar to P450 11B1, P450 11B2 also forms a complex with the Adx dimer via three lysine residues. We describe similarities and differences in our models of the P450 11B1·Adx2 and P450 11B2·Adx2 complexes with the structure of the P450 11A1-Adx fusion protein.

Evolutionary history of redox metal-binding domains across the tree of life.

Oxidoreductases mediate electron transfer (i.e., redox) reactions across the tree of life and ultimately facilitate the biologically driven fluxes of hydrogen, carbon, nitrogen, oxygen, and sulfur on Earth. The core enzymes responsible for these reactions are ancient, often small in size, and highly diverse in amino acid sequence, and many require specific transition metals in their active sites. Here we reconstruct the evolution of metal-binding domains in extant oxidoreductases using a flexible network approach and permissive profile alignments based on available microbial genome data. Our results suggest there were at least 10 independent origins of redox domain families. However, we also identified multiple ancient connections between Fe2S2- (adrenodoxin-like) and heme- (cytochrome c) binding domains. Our results suggest that these two iron-containing redox families had a single common ancestor that underwent duplication and divergence. The iron-containing protein family constitutes ∼50% of all metal-containing oxidoreductases and potentially catalyzed redox reactions in the Archean oceans. Heme-binding domains seem to be derived via modular evolutionary processes that ultimately form the backbone of redox reactions in both anaerobic and aerobic respiration and photosynthesis. The empirically discovered network allows us to peer into the ancient history of microbial metabolism on our planet.

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli.

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron-sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron-sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10mM HEPES buffer, pH7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at +0.4V (vs. Ag/AgCl) and a reduction peak at -0.3V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron-sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron-sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system.

In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1--the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.

Interactions of human mitochondrial Ferredoxin 1 (Adrenodoxin) by NMR; modulation by cytochrome P450 substrate and by truncation of the C-terminal tail.

Human Ferredoxin 1, also referred to as Adrenodoxin (Adx), is the sole electron carrier supporting the function of all seven mitochondrial cytochrome P450 (CYP) enzymes. Adx utilizes conserved negatively charged residues along its α-helix3 to interact with either the proximal surface of CYP enzymes or the binding surface of Adrendodoxin Reductase (AdR). However, in the oxidized state, Adx assumes a monomer-homodimer equilibrium that requires the presence of its unstructured C-terminal tail. Crystallographic structures of full-length human Adx dimers indicate that part of the binding surface necessary for its interactions with CYPs or with AdR is partially occluded by the dimer interface. In this study, protein NMR spectroscopy was used to interrogate the interactions between full-length (2-124) or truncated monomeric (2-108) human Adx and human CYP24A1 (with and without its vitamin-D substrate) as well as interactions with AdR. Here, monomeric Adx induced a similar pattern of peak broadening as that induced by addition of CYP24A1 substrate, consistent with a 1:1 Adx:CYP interaction as the functional complex. Additionally, removal of the C-terminal tail appears to enhance the interaction with AdR, despite removal of some of the AdR contacts in the tail region. This finding was also supported by an NMR competition assay. These findings suggest that the Adx dimers do not undergo meaningful interactions with either CYP or AdR, but may instead be responsible for regulating access to monomeric Adx. These conclusions are discussed in the context of a revised model of the Adx electron shuttle mechanism.

Mitochondrial [2Fe-2S] ferredoxins: new functions for old dogs.

Ferredoxins (FDXs) comprise a large family of iron-sulfur proteins that shuttle electrons from NADPH and FDX reductases into diverse biological processes. This review focuses on the structure, function and specificity of mitochondrial [2Fe-2S] FDXs that are related to bacterial FDXs due to their endosymbiotic inheritance. Their classical function in cytochrome P450-dependent steroid transformations was identified around 1960, and is exemplified by mammalian FDX1 (aka adrenodoxin). Thirty years later the essential function in cellular Fe/S protein biogenesis was discovered for the yeast mitochondrial FDX Yah1 that is additionally crucial for the formation of haem a and ubiquinone CoQ6 . In mammals, Fe/S protein biogenesis is exclusively performed by the FDX1 paralog FDX2, despite the high structural similarity of both proteins. Recently, additional and specific roles of human FDX1 in haem a and lipoyl cofactor biosyntheses were described. For lipoyl synthesis, FDX1 transfers electrons to the radical S-adenosyl methionine-dependent lipoyl synthase to kickstart its radical chain reaction. The high target specificity of the two mammalian FDXs is contained within small conserved sequence motifs, that upon swapping change the target selection of these electron donors.

Comparative biochemical characterization of mammalian-derived CYP11A1s with cholesterol side-chain cleavage activities.

Steroid drugs, the second largest class of pharmaceuticals after antibiotics, have shown significant anti-inflammatory, anti-allergic, and endocrine-regulating effects. A group of cytochrome P450 enzymes, namely, CYP11A1 isoenzymes from different organisms are capable of converting cholesterol into pregnenolone, which is a pivotal reaction in both steroid metabolism and (bio)synthetic network of steroid products. However, the low activity of CYP11A1s greatly restricts the industrial application of these cholesterol side-chain cleavage enzymes. Herein, we investigate ten CYP11A1 enzymes of different origins and in vitro characterize two CYP11A1s with a relatively higher expression level from Capra hircus and Sus scrofa, together with the CYP11A1s from Homo sapiens and Bos taurus as references. Towards five selected sterol substrates with different side chain structures, S. scrofa CYP11A1 displays relatively higher activities. Through redox partners combination screening, we reveal the optimal redox partner pair of S. scrofa adrenodoxin and C. hircus adrenodoxin reductase. Moreover, the semi-rational mutagenesis for the active sites and substrate entrance channels of human and bovine CYP11A1s is performed based on comparative analysis of their crystal structures. The mutant mBtCYP11A1-Q377A derived from mature B. taurus CYP11A1 shows a 1.46 times higher activity than the wild type enzyme. These results not only demonstrate the tunability of the highly conserved CYP11A1 isoenzymes, but also lay a foundation for the following engineering efforts on these industrially relevant P450 enzymes.

Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins.

Adrenodoxin is probably the best characterized member of the vertebrate-type [2Fe-2S]-cluster ferredoxins. It has been in the spotlight of scientific interest for many years due to its essential role in mammalian steroid hormone biosynthesis, where it acts as electron mediator between the NADPH-dependent adrenodoxin reductase and several mitochondrial cytochromes P450. In this review we will focus on the present knowledge about protein-protein recognition in the mitochondrial cytochrome P450 system and the modulation of the electron transfer between Adx and its redox partners, AdR and CYP(s). We also intend to point out the potential biotechnological applications of Adx as a versatile electron donor to different cytochromes P450, both in vitro and in vivo. Finally we will address the comparison between the mammalian cytochrome P450-associated adrenodoxin and ferredoxins involved in iron-sulfur-cluster biosynthesis. Despite their different functions, these proteins display an amazing similarity regarding their primary sequence, tertiary structure and biophysical features.

Adrenodoxin--a versatile ferredoxin.

Mammalian adrenodoxin (Adx) has been known for many years as an essential electron mediator in mitochondrial cytochrome P450 systems. Because of its ability to support several cytochrome P450 enzymes, it is involved not only in adrenal steroid hormone biosynthesis but also in vitamin D and bile acid metabolism. Recently, Adx is increasingly gaining attention because of its potential for pharmaceutical industry and biotechnology. With human cytochromes P450 becoming important drug targets, suitable Adx-based screening systems have to be developed to test putative new drugs. Moreover, in artificial systems, Adx has been shown to functionally interact with diverse bacterial cytochromes P450 catalyzing a variety of chemically interesting reactions. Putative biotechnological applications of such Adx-containing reconstituted systems are discussed.

[Adrenodoxins and their role in the cytochrome P450 systems]. / Adrenodoksiny i ikh rol' v sistemakh tsitokhroma R450.

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻6 M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻8 M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.

Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition.

Human cytochrome P450 11B1 (CYP11B1) generation of the major glucocorticoid cortisol requires two electrons delivered sequentially by the iron­sulfur protein adrenodoxin. While the expected adrenodoxin binding site is on the opposite side of the heme and 15-20 Å away, evidence is provided that adrenodoxin allosterically impacts CYP11B1 ligand binding and catalysis. The presence of adrenodoxin both decreases the dissociation constant (Kd) for substrate binding and increases the proportion of substrate that is bound at saturation. Adrenodoxin additionally decreases the Michaelis-Menten constant for the native substrate. Similar studies with several inhibitors also demonstrate the ability of adrenodoxin to modulate inhibition (IC50 values). Somewhat similar allosterism has recently been observed for the closely related CYP11B2/aldosterone synthase, but there are several marked differences in adrenodoxin effects on the two CYP11B enzymes. Comparison of the sequences and structures of these two CYP11B enzymes helps identify regions likely responsible for the functional differences. The allosteric effects of adrenodoxin on CYP11B enzymes underscore the importance of considering P450/redox partner interactions when evaluating new inhibitors.

Molecular characterization of a class I P450 electron transfer system from Novosphingobium aromaticivorans DSM12444.

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (k(cat) = 39-91 s(-1)) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity.

Substrate-induced modulation of protein-protein interactions within human mitochondrial cytochrome P450-dependent system.

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11 s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s-Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 - Adx complex formation without significant effect on dissociation (koff decreased ∼1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 - Adx and CYP11B2 - Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

A solution model of the complex formed by adrenodoxin and adrenodoxin reductase determined by paramagnetic NMR spectroscopy.

Lanthanide tags offer the opportunity to retrieve long-range distance information from NMR experiments that can be used to guide protein docking. To determine whether sufficient restraints can be retrieved for proteins with low solubility and availability, Ln tags were applied in the study of the 65 kDa membrane-associated protein complex formed by the electron carrier adrenodoxin and its electron donor, adrenodoxin reductase. The reductase is only monomeric at low concentration, and the paramagnetic iron-sulfur cluster of adrenodoxin broadens many of the resonances of nuclei in the interface. Guided by the paramagnetic restraints obtained using two Ln-tag attachment sites, protein docking yields a cluster of solutions with an rmsd of 3.2 A. The mean structure is close to the crystal structure of the cross-linked complex, with an rmsd of 4.0 A. It is concluded that with the application of Ln tags paramagnetic NMR restraints for structure determination can be retrieved even for difficult, low-concentration protein complexes.