Specificity of the Redox Complex between Cytochrome P450 24A1 and Adrenodoxin Relies on Carbon-25 Hydroxylation of Vitamin-D Substrate.

Metabolic deactivation of 1,25(OH)2D3 is initiated by modification of the vitamin-D side chain, as carried out by the mitochondrial cytochrome P450 24A1 (CYP24A1). In addition to its role in vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. CYP24A1 function relies on electron transfer from the soluble ferredoxin protein adrenodoxin (Adx). Recent structural evidence suggests that regioselectivity of the CYP24A1 reaction may correlate with distinct modes of Adx recognition. Here we used nuclear magnetic resonance (NMR) spectroscopy to monitor the structure of 15N-labeled full-length Adx from rat while forming the complex with rat CYP24A1 in the ligand-free state or bound to either 1,25(OH)2D3 or the vitamin-D supplement 1α(OH)D3. Although both vitamin-D ligands were found to induce a reduction in overall NMR peak broadening, thereby suggesting ligand-induced disruption of the complex, a crosslinking analysis suggested that ligand does not have a significant effect on the relative association affinities of the redox complexes. However, a key finding is that, whereas the presence of primary CYP24A1 substrate was found to induce NMR peak broadening focused on the putative recognition site α-helix 3 of rat adrenodoxin, the interaction in the presence of 1α(OH)D3, which is lacking the carbon-25 hydroxyl, results in disruption of the NMR peak broadening pattern, thus indicating a ligand-induced nonspecific protein interaction. These findings provide a structural basis for the poor substrate turnover of side-chain-modified vitamin-D analogs, while also confirming that specificity of the CYP24A1-ligand interaction influences specificity of CYP24A1-Adx recognition. SIGNIFICANCE STATEMENT: Mitochondrial cytochrome P450 enzymes, such as CYP24A1 responsible for catabolizing vitamin-D and its analogs, rely on a protein-protein interaction with a ferredoxin in order to receive delivery of the electrons required for catalysis. In this study, we demonstrate that this protein interaction is influenced by the enzyme-ligand interaction that precedes it. Specifically, vitamin-D missing carbon-25 hydroxylation binds the enzyme active site with high affinity but results in a loss of P450-ferredoxin binding specificity.

Kinetic and optical biosensor study of adrenodoxin mutant AdxS112W displaying an enhanced interaction towards the cholesterol side chain cleavage enzyme (CYP11A1).

In mammals, steroid hormones are synthesized from cholesterol that is metabolized by the mitochondrial CYP11A1 system leading to pregnenolone. The reduction equivalents for this reaction are provided by NADPH, via a small electron transfer chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). The reaction partners are involved in a series of transient interactions to realize the electron transfer from NADPH to CYP11A1. Here, we compared the ionic strength effect on the AdR/Adx and Adx/CYP11A1 interactions for wild-type Adx and mutant AdxS112W. Using surface plasmon resonance measurements, stopped flow kinetic investigations and analyses of the product formation, we were able to obtain new insights into the mechanism of these interactions. The replacement of serine 112 by tryptophan was demonstrated to lead to a dramatically decreased k (off) rate of the Adx/CYP11A1 complex, resulting in a four-fold decreased K (d) value and indicating a much higher stability of the complex involving the mutant. Stopped flow analysis at various ionic strengths and in different mixing modes revealed that the binding of reduced Adx to CYP11A1 seems to display the limiting step for electron transfer to CYP11A1 with pre-reduced AdxS112W being much more efficient than wild-type Adx. Finally, the dramatic increase in pregnenolone formation at higher ionic strength using the mutant demonstrates that the interaction of CYP11A1 with Adx is the rate-limiting step in substrate conversion and that hydrophobic interactions may considerably improve this interaction and the efficiency of product formation. The data are discussed using published structural data of the complexes.

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1).

CYP27B1 catalyzes the 1alpha-hydroxylation of 25-hydroxyvitamin D3 to 1alpha,25-dihydroxyvitamin D3, the hormonally active form of vitamin D3. To further characterize mouse CYP27B1, it was expressed in Escherichia coli, purified and its activity measured on substrates incorporated into phospholipid vesicles, which served as a model of the inner mitochondrial membrane. 25-Hydroxyvitamin D3 and 25-hydroxyvitamin D2 in vesicles underwent 1alpha-hydroxylation with similar kinetics, the catalytic rate constants (k(cat)) were 41 and 48mol/min/mol P450, respectively, while K(m) values were 5.9 and 4.6mmol/mol phospholipid, respectively. CYP27B1 showed inhibition when substrate concentrations in the membrane were greater than 4 times K(m), more pronounced with 25-hydroxyvitamin D3 than 25-hydroxyvitamin D2. Higher catalytic efficiency was seen in vesicles prepared from dioleoyl phosphatidylcholine and cardiolipin than for dimyristoyl phosphatidylcholine vesicles. CYP27B1 also catalyzed 1alpha-hydroxylation of vesicle-associated 24R,25-dihydroxyvitamin D3 and 20-hydroxyvitamin D3, and 25-hydroxylation of 1alpha-hydroxyvitamin D3 and 1alpha-hydroxyvitamin D2, but with much lower efficiency than for 25(OH)D3. This study shows that CYP27B1 can hydroxylate 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 associated with phospholipid membranes with the highest activity yet reported for the enzyme. The expressed enzyme has low activity at higher concentrations of 25-hydroxyvitamin D in membranes, revealing that substrate inhibition may contribute to the regulation of the activity of this enzyme.

The interaction domain of the redox protein adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor adrenodoxin reductase.

Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in electron transfer reactions in the steroid hormone biosynthesis of mammals. In this study, we deleted the sequence coding for the complete interaction domain in the Adx cDNA. The expressed recombinant protein consists of the amino acids 1-60, followed by the residues 89-128, and represents only the core domain of Adx (Adx-cd) but still incorporates the [2Fe-2S] cluster. Adx-cd accepts electrons from its natural redox partner, adrenodoxin reductase (AdR), and forms an individual complex with this NADPH-dependent flavoprotein. In contrast, formation of a complex with the natural electron acceptor, CYP11A1, as well as electron transfer to this steroid hydroxylase is prevented. By an electrostatic and van der Waals energy minimization procedure, complexes between AdR and Adx-cd have been proposed which have binding areas different from the native complex. Electron transport remains possible, despite longer electron transfer pathways.

Unfolding and conformational studies on bovine adrenodoxin probed by engineered intrinsic tryptophan fluorescence.

An intrinsic steady-state fluorescent system for bovine adrenodoxin has been developed to study the protein structure in solution and the processes involved in protein unfolding. Since mature Adx contains no natural Trp residue as internal probe, all of the aromatic amino acids, tyrosine at position 82 and four phenylalanines at positions 11, 43, 59 and 64, were at each case replaced by tryptophan. The resulting single tryptophan containing mutants kept their biological function compared with the wild type. Molecular modeling studies verify thermal unfolding experiments which point to a dramatically reduced stability caused by steric hindrance only for mutant F59W. Fluorescence spectra, Stern-Volmer quenching constants, and fluorescence energy transfer calculations indicated the analyzed positions to be situated in solution in the same immediate environment as in the crystal structure. Unfolding experiments with Gdn-HCl and time-resolved stopped-flow measurements provide evidence for differential stability and a chronologically ordered unfolding mechanism of the different fluorescence probe positions in the protein.

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.

Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2Fe-2S] cluster.

The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions. The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.

Comparative structural-functional characterization of recombinant and natural adrenodoxin. Interaction with cytochrome P450scc.

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.

GroEL-assisted and -unassisted refolding of mature and precursor adrenodoxin: the role of the precursor sequence.

We have performed refolding studies on a [2Fe-2S] protein, adrenodoxin (Adx), and its precursor form, preadrenodoxin. In vitro, mature Adx is expressed as a soluble active form in Escherichia coli, but precursor Adx is expressed in inclusion bodies. Both mature and precursor Adx refolded spontaneously from their denatured forms and the recovery levels of enzyme activities were 40 and 37% for mature and precursor Adx, respectively. Furthermore, the interaction between GroEL- and Gdn-HCl-denatured mature and precursor forms was investigated. In the case of mature Adx, the activity was increased in the presence of either GroEL, GroES, or bovine serum albumin and the refolding of mature Adx is a nonspecific process. However, the GroEL-mediated reaction is specific for precursor Adx under the experimental conditions used here. A higher electron transfer activity is obtained after ATP addition to the GroEL-containing refolding mixture, and GroEL-precursor complexes were found by gel chromatography studies. Our observation suggests that the small single-domain protein Adx (mature form) folded independently of the chaperonin GroEL. The contribution of the chaperonin complexes to the folding is toward the aggregation-sensitive precursor Adx, which in vitro folded 1.3- to 1.4-fold slower than mature Adx. This demonstrates that the presequence is responsible for the formation of inclusion bodies and for the in vitro recognition motif for GroEL binding.

Overproduction in Escherichia coli and characterization of the precise mature form of rat adrenodoxin.

The mature form of rat adrenodoxin (Ad) was purified from a heterologous direct expression system in Escherichia coli with a yield of 56 mg/l culture. The purified Ad showed a A414/A280 ratio of 0.91 and the sequence of 10 amino terminal residues was identical with that of authentic rat Ad. By Time of Flight/Mass spectrometry, the molecular mass of purified Ad was identical to that calculated from the cDNA sequence and the carboxy terminal residue was estimated to be Ser, which was also as expected from the cDNA. These results indicate that the purified recombinant Ad is a precise mature form. In measurements of NADPH-cytochrome c reductase activity reconstituted with bovine adrenodoxin reductase (AdR), the apparent Km value for rat Ad was 46.9+/-2.5 nM, indicating a somewhat lower affinity for rat Ad to bovine AdR than for bovine Ad. On the other hand, the spectral Kd value for rat Ad to bovine cytochrome P-450scc was 0.46+/-0.05 microM, a value which was almost identical with that of the bovine counterpart.

Langmuir-Blodgett films of cytochrome P450scc and its complex with adrenodoxin.

Langmuir-Blodgett films prepared from cytochrome P450scc and its complex with adrenodoxin have been prepared and studied. Adrenodoxin was preliminarily selectively modified with fluorescein isothiocyanate and the effect of this modification on the interaction with cytochrome P450scc was studied. Using selectively modified adrenodoxin the ratio of the proteins in the film was found to be 1 mole of adrenodoxin per 2 moles of cytochrome P450scc. Langmuir-Blodgett films were also prepared from adrenodoxin-reductase and it was shown that this flavoprotein is transferred to the substrate as an apo-protein. It is also shown that the adrenodoxin-binding region of cytochrome P450scc is exposed to the subphase under all pressures in the interval studied. The relationship between the orientation of cytochrome P450scc-adrenodoxin complex in monolayers on the water-air interface and the pressure produced at the interface at the moment of monolayer formation was found. Our data are in excellent accordance with ideas on the molecular organization of cytochrome P450scc in the inner adrenocortical membrane and allows the use of this approach to model membrane structures containing cytochrome P450.

Different effects of carboxy-terminal deletion in the adrenodoxin molecule on cytochrome c and acetylated cytochrome c reductions.

In immunoblotting analysis using a rabbit antibody to bovine adrenodoxin, the total proteins of the bovine adrenal cortex gave two bands, suggesting the presence of two forms of adrenodoxin in vivo: full-length and carboxy-terminal deleted adrenodoxins. To examine the effect of the carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrenodoxin were constructed. The wild type [Ad(2-128)] and carboxy-terminal deleted [Ad(2-114) and Ad(2-112)] recombinant adrenodoxins expressed in Escherichia coli were purified to give a single band on SDS-PAGE. They showed an A414/A276 value of 0.92. In an NADPH-cytochrome c reduction assay, the Km values for cytochrome c in the reconstituted system with AD(2-128), Ad(2-114) and Ad(2-112) were 39, 235 and 618 mM, respectively. The Vmax values were 638, 700 and 898 mol/min/mol flavin, respectively. In an NADPH-acetylated cytochrome c reduction assay, the maximum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maximum activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values were 4-times higher than that of Ad(2-128). In the presence of 100 mM NaCl, the Km values for acetylated cytochrome c in the system reconstituted with Ad(2-128), Ad(2-114) and Ad(2-112) were 220, 33 and 22 microM, respectively. The Vmax values were 352, 305 and 382 mol/min/mol flavin, respectively. These results indicate that the effects of the carboxy-terminal deletion of adrenodoxin on NADPH-cytochrome c and acetylated cytochrome c reductions are different; the carboxy-terminal region (residues 113-128) of adrenodoxin largely contributes to the binding with cytochrome c but disturbs the binding with acetylated cytochrome c.

Pyridoxal phosphate induced association reactions of adrenodoxin and adrenodoxin-reductase.

Pyridoxal phosphate, cofactor of several enzymes, possesses linking properties to induce oligomerization of identical proteins such as adrenodoxin-reductase or adrenodoxin. The capability to get such self-assemblies is slightly lower than that for obtaining the heterologous complex between adrenodoxin-reductase and adrenodoxin which was found to be essential for electron transfer in the cytochrome P450 system. The influence of pyridoxal phosphate on the complex formation between adrenodoxin-reductase and adrenodoxin as well as the oligomerization reaction of the isolated proteins and possible consequences is discussed.

Bovine adrenodoxin--a mitochondrial iron-sulphur protein--binds to chaperonin GroEL.

The interaction of bovine adrenodoxin with the chaperonin GroEL was investigated using sucrose density centrifugation and analytical ultracentrifugation. It could be clearly established that denatured mature adrenodoxin comigrated in a sucrose density gradient with the GroEL oligomer, indicating that a complex had been formed. Up to 2 moles of adrenodoxin/mol GroEL can be bound. From the partial concentrations, association constants of 4.3 x 10(5) M-1 for the first adrenodoxin molecule and of 1.08 x 10(5) M-1 for the second molecule to the complex, respectively, were calculated. Upon addition of the cochaperonin GroES and Mg-ATP to the adrenodoxin-GroEL complex, adrenodoxin was released, indicating a specific binding between GroEL and adrenodoxin.

Purification and characterization of rat adrenodoxin.

Adrenodoxin was purified from the rat adrenal gland. The A414/A280 value of the purified rat adrenodoxin was 0.90 and the oxidized spectrum showed absorption maxima at 320, 414 and 455 nm, similar to those of bovine adrenodoxin. On SDS-PAGE, the rat adrenodoxin showed a single band with a molecular mass of 11.2 kDa, while the apparent molecular mass by gel filtration through Sephadex G-75 equilibrated with 10 mM K-phosphate (pH 7.5) was 27 kDa. In the reconstituted system, Vmax of NADPH-cytochrome c reduction activity and the Km for the rat adrenodoxin were much the same as those for recombinant bovine adrenodoxin. In the case of cholesterol side-chain cleavage activity, however, these values of the rat adrenodoxin were about half of those of the bovine adrenodoxin. The CD spectrum of the rat adrenodoxin was similar to that of the bovine adrenodoxin but showed a significantly lower ellipticity value in the 195-205 nm region than that of the bovine adrenodoxin. The structural differences may possibly explain differences in the enzymic properties between rat and bovine adrenodoxins.

Catalytic properties of cytochrome P-450scc purified from the human placenta: comparison to bovine cytochrome P-450scc.

Cytochrome P-450scc was purified from the human placenta by extraction of mitochondria with cholate and Emulgen 911, chromatography on phenyl-Sepharose and DEAE-Sephacel, and ammonium sulphate fractionation. The catalytic properties of the purified human cytochrome P-450scc were analysed in Tween-20 micelles and compared to those of bovine adrenal cytochrome P-450scc analysed in the same system. Both enzymes had the same Km for cholesterol and were stimulated by cardiolipin when the cholesterol concentration was subsaturating. Examination of the rates of pregnenolone synthesis from 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol and 20 alpha, 22R-dihydroxycholesterol by human and bovine cytochromes P-450scc revealed that the first hydroxylation (22R position) was rate-limiting for both in Tween-20 micelles. The rate of the 22R-hydroxylation was further decreased when a 20 alpha-hydroxyl group was already present on the cholesterol side-chain. The second hydroxylation occurred at about the same rate as the third hydroxylation for both enzymes. The rate of side-chain cleavage of 25-hydroxycholesterol by human cytochrome P-450scc in Tween-20 micelles was low, the highest rate being about 1% of the Vmax for cholesterol. Substrate inhibition was seen with high concentrations of 25-hydroxycholesterol. Conversion of 25-hydroxycholesterol to pregnenolone was accompanied by a build-up of products with intact side-chains, which were probably intermediates of the reaction. Side-chain cleavage of 25-hydroxycholesterol by bovine cytochrome P-450scc showed similar characteristics to the human enzyme, except that the highest velocity observed was approx. 25% of the Vmax for cholesterol. Rates of cleavage of 25-hydroxycholesterol by both enzymes were higher in dioleoylphosphatidylcholine vesicles than in Tween-20, but were still well below the Vmax for cholesterol and showed substrate inhibition. This study shows that there is close similarity in catalytic properties between human and bovine cytochromes P-450scc which suggests that the active site of the cytochrome is highly conserved.

Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands.

Expression systems for adrenodoxin into the periplasm and the cytoplasm of E. coli have been developed as a prerequisite for site-directed mutagenesis studies. In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level. To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines. From these mutants, only C95S forms a functionally active holoprotein. Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions.

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met1), Ad(Met-1), Ad(Met-12), and Ad(Met6). The shoulder numbers represent the site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met-1), were purified from the cell lysate and the ratios of A414-to-A276 of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450SCC reduction catalyzed by Ad(Met6) was about 60% of that by Ad(Met1), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450SCC would need to be considered.

Characterization of adrenodoxin precursor expressed in Escherichia coli.

The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP.

[Comparative study of bovine adrenodoxin and renorenodoxin]. / Sravnitel'noe izuchenie adrenodoksina i renoredoksina byka.

The ferredoxin from bovine renal mitochondria (renoredoxin) has been obtained in a highly purified state. The A415/A280 ratio of the purified renoredoxin is 0.84. The absorption spectrum of renoredoxin was shown to be identical to that of bovine adrenodoxin. Two forms of renoredoxin (Mr 14200 and 13300) were detected by using polyacrylamide gel electrophoresis. These forms exhibit a very similar immunologic cross-reactivity with polyclonal antibodies to adrenodoxin. The N-terminal amino acid sequence of renal ferredoxin was shown to be identical to that of adrenodoxin; the C-terminal sequences of both ferredoxins undergo a similar post-translational proteolytic modification. The amino acid composition of ferredoxins are also very close. Renal ferredoxin can be replaced by adrenodoxin in reconstituted systems from bovine adrenal cortex mitochondria which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone.