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At least 19 recordsLinked to original sources
PubMed1984

Adrenodoxin interaction with adrenodoxin reductase and cytochrome P-450scc. Cross-linking of protein complexes and effects of adrenodoxin modification by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Modification of the three carboxyl groups on adrenodoxin using a water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) caused a weakening of the binding of this iron-sulfur protein to both its electron donor protein, adrenodoxin reductase, and its electron acceptor protein, cytochrome P-450scc. Based upon the proximity of the modified groups, the site on adrenodoxin for interaction with the other two proteins is likely to be either identical or highly overlapping, and formation of a ternary complex among the proteins is precluded. Upon incubation of adrenodoxin and either adrenodoxin reductase or cytochrome P-450 plus the carbodiimide (1:1), covalently cross-linked species were formed. When all three proteins were incubated with the cross-linker, only the binary complexes were formed, and no higher order (e.g. 1:1:1 or 1:2:1) complexes were seen. These studies indicate that adrenodoxin forms exclusive binary complexes with its electron transfer partner proteins, and thus provide a physical explanation for the proposed role of adrenodoxin as a mobile electron shuttle between NADPH-adrenodoxin reductase and cytochrome P-450scc.

Adrenodoxin↗
PubMed1983

In vitro synthesis of NADPH-adrenodoxin reductase and adrenodoxin of bovine adrenal cortex, and the existence of a large precursor of adrenodoxin.

Cytoplasmic free and bound polysomes isolated from bovine adrenal cortex were used to program protein synthesis in rat liver cell sap (175,000 X g soluble material) and wheat germ lysate S-175 (175,000 X g soluble material) systems. Both free and bound polysomes were translated with high fidelity and equal efficiency in the cell-free systems. Synthesis of adrenodoxin reductase (AdR) and adrenodoxin (Ad) in the cell-free systems was determined by the immunoprecipitation of the translation products using monospecific antibodies, and we found that AdR was synthesized by free polysomes whereas Ad was synthesized by both free and bound polysomes as a large precursor (pAd) having a molecular weight of approximately 22,000 daltons, which is about 10,000 daltons larger than mature Ad. The existence of a large precursor of Ad was also confirmed by its synthesis in a cell-free system programmed by total RNA isolated from bovine adrenal cortex. Bovine adrenal cortex mitochondria imported in vitro-synthesized AdR and pAd to the inside of the organelle in the absence of protein synthesis in a cell-free system, and processed pAd to the mature form during the import. These results suggest that cytoplasmically synthesized AdR and Ad are translocated posttranslationally into the matrix of mitochondria in adrenal cortex cells.

Adrenal Cortex↗
PubMed2001

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.

Adrenodoxin↗
PubMed1987

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc.

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. Förster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.

Adrenodoxin↗
PubMed1978

Crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from pig adrenocortical mitochondria. Essential histidyl and cysteinyl residues of the NADPH-binding site and environment of the adrenodoxin-binding site.

Pig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49,000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.

Adrenal Cortex↗
PubMed1983

The effect of pH on the formal reduction potential of adrenodoxin in the presence and absence of adrenodoxin reductase: the implication in the electron transfer mechanism.

We have investigated the formal reduction potentials (E degrees') of adrenodoxin with and without adrenodoxin reductase in order to elucidate the mechanism of electron transfer from adrenodoxin reductase (a flavoprotein) to adrenodoxin (an iron-sulfur protein). It was found by our spectropotentiostatic method that adrenodoxin showed no variation of E degrees' at different pH's in the absence of adrenodoxin reductase. The average E degrees' was -252 +/- 2 mV in the pH range between 6.0 and 8.3. In the presence of adrenodoxin reductase, adrenodoxin exhibited, on the other hand, a pH dependence of E degrees' at pH higher than 7.2 with a slope of -59 mV per pH unit: Adrenodoxin molecule possesses one protonation site with a pKa of 7.2. Cyclic voltammograms of adrenodoxin additionally revealed that the reoxidation reaction of reduced adrenodoxin is very slow in the absence of adrenodoxin reductase, but that it is readily reoxidized in the presence of adrenodoxin reductase.

Adrenal Cortex↗
PubMed1991

Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase.

Three histidine residues of bovine adrenodoxin, His-10, His-56, and His-62, were modified with diethyl pyrocarbonate. The order of the modification among the three histidines were monitored by measuring the proton NMR spectra. The modified adrenodoxin exhibited reduced affinity for adrenodoxin reductase as determined in cytochrome c reductase activity. In the presence of cholesterol, the modified adrenodoxin induced a high spin form of cytochrome P-450scc on complex formation in the same manner as native adrenodoxin. The spectral titration showed that adrenodoxin modified with diethyl pyrocarbonate exhibited a 5-fold higher Kd value than that of native adrenodoxin. These effects of the modification of adrenodoxin on the affinities for the redox partners were not proportional to the number of modified histidines determined by the optical absorbance change at 240 nm. Modification of adrenodoxin up to 2 histidine residues did not affect the affinity for the redox partners, but further modification on the third one resulted in an increase of apparent Km in cytochrome c reductase activity by 2-fold and of Kd for cytochrome P-450scc by 5-fold. The 1H NMR spectra of the modified adrenodoxin unequivocally demonstrated that histidine residues at His-10 and His-62 reacted more readily with diethyl pyrocarbonate than His-56 did, indicating that modification of His-56 was responsible for the reduction of binding affinities of adrenodoxin for redox partners. These results are consistent with the proposal that the residue of His-56 in adrenodoxin has an essential role in the electron transfer mechanism where adrenodoxin functions as a mobile shuttle.

Adrenodoxin↗
PubMed1977

Adrenodoxin reductase and adrenodoxin. Mechanisms of reduction of ferricyanide and cytochrome c.

Adrenodoxin reductase, the flavoprotein moiety of the adrenal cortex mitochondrial steroid hydroxylating system, participates in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these 1-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and 2-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction. For cytochrome c reduction, the adrenodoxin reductase-adrenodoxin 1:1 complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251, 7545-7550). Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k approximately or equal to 4.6 s-1), but only slowly with a second cytochrome c (k = 0.1 to 0.3 s-1). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase-adrenodoxin complex appears to cycle between 1- and 3-electron reduced states, via an intermediate 2-electron-containing form produced by reoxidation by cytochrome (Fig. 10). For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center...

Adrenal Cortex↗
PubMed1985

Cross-linking studies of steroidogenic electron transfer: covalent complex of adrenodoxin reductase with adrenodoxin.

Bifunctional reagents 3,3'-dithiobis(succinimidyl propionate), 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide and N-succinimidyl 3-(2-pyridyldithio)propionate have been used in an attempt to study molecular organization and covalent cross-linking of adrenodoxin reductase with adrenodoxin, the components of steroidogenic electron transfer system in bovine adrenocortical mitochondria. There was no cross-linking of individual proteins by the bifunctional reagents used, except for adrenodoxin cross-linking with water-soluble carbodiimide. Substantial cross-linking of adrenodoxin reductase with adrenodoxin was observed when water-soluble carbodiimide was used as cross-linking reagent. However, the cross-linked complex failed to transfer electrons. Significant amounts of the functional cross-linked complex (up to 42%) were observed when the proteins were cross-linked with N-succinimidyl 3-(2-pyridyldithio)propionate. Using gel filtration, ion-exchange chromatography and affinity chromatography on adrenodoxin-Sepharose, the complex was obtained in a highly purified form. In the presence of cytochrome P-450scc or cytochrome c, the cross-linked complex of adrenodoxin reductase with adrenodoxin was active in electron transfer from NADPH to heme proteins. The data obtained indicate that there are distinct binding sites on the adrenodoxin molecule responsible for the adrenodoxin reductase and cytochrome P-450scc binding, which suggests that steroidogenic electron transfer may be realized in an organized complex.

Adrenal Cortex↗
PubMed2000

Construction and characterization of a catalytic fusion protein system: P-450(11beta)-adrenodoxin reductase-adrenodoxin.

Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step procedures. The most critical step is the 11beta-hydroxylation of 11-deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11beta). Various fusion constructs of P-450(11beta) with its electron transfer components, adrenodoxin and adrenodoxin reductase, were produced by cDNA manipulation and were successfully expressed in COS-1 cells from which the hydroxylation activities were assayed. It was demonstrated that the fusion protein required both adrenodoxin reductase and adrenodoxin for its activity and was not able to receive electrons from an external source. The fusion protein with all three components had less activity than P-450(11beta) alone, receiving electrons from coexpressed or internal electron transfer components. The activities of the fusion proteins were determined mainly by the fusion sequence. The fusion protein with a sequence of P-450(11beta)-adrenodoxin reductase-adrenodoxin was more active than that of P-450(11beta)-adrenodoxin-adrenodoxin reductase, 1.5- and 3-fold for bovine and human P-450(11beta), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11beta)-adrenodoxin reductase-adrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusion protein obtained here can serve as a model for the investigation of electron transfer in P-450 systems and is of potential importance for biotechnological steroid production.

Adrenodoxin↗
PubMed1999

An additional electrostatic interaction between adrenodoxin and P450c27 (CYP27A1) results in tighter binding than between adrenodoxin and p450scc (CYP11A1).

Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene). To investigate the basis of the approximately 30-fold difference in adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified. Mutations of one set were within the putative adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding adrenodoxin. The second set included mutations within a sequence aligning with the "meander region" of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62). Mutation of the P450c27 conserved lysines (K354A and K358A) led to a approximately 20-fold increase in apparent Ks for adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for adrenodoxin binding. Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for adrenodoxin approximately 20-fold. This residue is predicted to be located in the meander region. A triple K354A/K358A/R418S mutation profoundly reduced adrenodoxin binding. Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for adrenodoxin interaction.

Adrenodoxin↗
PubMed1976

Studies on nitrotyrosine-82 and aminotyrosine-82 derivatives of adrenodoxin. Effects of chemical modification on the complex formation with adrenodoxin reductase.

The coordination structure of the iron-sulfur center of the nitrotyrosine and the aminotyrosine derivates of bovine adrenodoxin was investigated by electron paramagnetic resonance spectroscopy. The reduced form of both modified samples exhibited signals identical with those for the native protein at g= 1.94 and g=2.01. From these results together with optical absorption and chemical analyses, it was concluded that the coordination structure of the iron-sulfur chromophore for both the derivatives was identical with the binuclear tetrahedral structure of native adrenodoxin. The configuration of the iron-binding area in nitro- and amino-adrenodoxin was studied by ovserving the circular dichroism spectra between 350 and 600 nm. The maxima for the nitro or amino derivatives were all identical with those for the native protein but different in the magnitude of their molar ellipticity. The molar ellipticities at 440 nm were 45.8 X 10(3), 14.5 X 10(3), and 9.5 X 10(6) deg cm2 per mol of iron for native adrenodoxin, nitro or amino derivative, respectively. These results suggest that the chemical modification of the tyrosine residue causes a conformational change in the iron-binding area. We have previously reported that the enzymatic activities of these reconstituted nitro and amino derivatives toware cytochrome c reduction in the presence of adrenodoxin reductase and reduced nicotinamide adenine dinucleotide phosphate were 19 and 7% of native adrenodoxin, respectively. The cytochrome c reductase activities of nitro- and aminoadrenodixin were drastically affected by the ionic strength of the assay medium, as found in native adrenodoxin. Fluorometric titration of the reductase with aminoadrenodoxin revealed that aminoadrenodoxin forms a 1:1 molar complex with the reductase. These results suggest that both the nitro and amino derivatives form a complex with the reductase. The dissociation constants of nitro- and aminoadrenodoxin for the reductase were 6.1 X 10(-7)M and 3.3 X 10(-7) M at mu = 0.04 and 1.9 X 10(-6) M and 2.0 X 10(-6) M at mu = 0.20, respectively. Comparison of these values with those of native adrenodoxin (approximately 10(-9) M at mu = 0.04 and 2.2 X 10(-7) M at mu = 0.20) suggests that an increase in the dissociation constant for the reductase is responsible for the decreased electron transferring activity of the modified adrenodoxins.

Adrenodoxin↗
PubMed1979

Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc.

Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.

Adrenal Cortex↗
PubMed1989

Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal 5'-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc.

Covalent modification of cytochrome P-450scc (purified from bovine adrenocortical mitochondria) with pyridoxal 5'-phosphate (PLP) was found to cause inhibition of the electron-accepting ability of this enzyme from its physiological electron donor, adrenodoxin, without conversion to the "P-420" form. Reaction conditions leading to the modification level of 0.82 and 2.85 PLP-Lys residues per cytochrome P-450scc molecule resulted in 60% and 98% inhibition, respectively, of electron-transfer rate from adrenodoxin to cytochrome P-450scc (with beta-NADPH as an electron donor via NADPH-adrenodoxin reductase and with phenyl isocyanide as the exogenous heme ligand of the cytochrome). It was found that covalent PLP modification caused a drastic decrease of cholesterol side-chain cleavage activity when the cholesterol side-chain cleavage enzyme system was reconstituted with native (or PLP-modified) cytochrome P-450scc, adrenodoxin, and NADPH-adrenodoxin reductase. Approximately 60% of the original enzymatic activity of cytochrome P-450scc was protected against inactivation by covalent PLP modification when 20% mole excess adrenodoxin was included during incubation with PLP. Binding affinity of substrate (cholesterol) to cytochrome P-450scc was found to be increased slightly upon covalent modification with PLP by analyzing a substrate-induced spectral change. The interaction of adrenodoxin with cytochrome P-450scc in the absence of substrate (cholesterol) was analyzed by difference absorption spectroscopy with a four-cuvette assembly, and the apparent dissociation constant (Ks) for adrenodoxin binding was found to be increased from 0.38 microM (native) to 33 microM (covalently PLP modified).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1976

Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochonria. I. Physicochemical properties of holo- and apo-NADPH-adrenodoxin reductase and interaction between non-heme iron proteins and the reductase.

A crystalline NADPH-adrenodoxin reductase was obtained from bovine adrenocortical mitochondria and its properties were investigated. Its molecular weights and isoelectric point were estimated to be 51 000 and 5.4, respectively. Amino acid and sugar contents and the interaction between the apo-reductase and flavin of NADPH-adrenodoxin reductase were investigated. Formation of a complex of bovine NADPH-adrenodoxin reductase with adrenodoxin, its apoadrenodoxin, or other non-heme iron proteins caused quenching of fluorescence of the tryptophanyl residue and bound FAD of the NADPH-adrenodoxin reductase. The results obatined suggest that adrenodoxin and apoadrenodoxin bind functionally to a site close to the tryptophanyl residue and the bound FAD of the reductase. The circular dichroism spectrum of oxidized NADPH-adrenodoxin reductase was measured in the ultraviolet and visible regions. This spectrum showed negative absorption in the visible region and was not appreciably influenced in either the ultraviolet or visible region by formation of a complex with adrenodoxin or apoadrenodoxin.

Adrenal Cortex↗
PubMed2001

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.

Adrenodoxin↗