Studies on NO2-Tyr82and NH2-Tyr82 derivatives of adrenodoxin. Effects of chemical modification on electron transferring activity.

Bovine apoadrenodoxin was treated with tetranitromethane to introduce a nitro group into the tyrosyl residue at position 82 of this protein. The degrees of nitration under the best conditions were estimated to be 90% and nearly 100% on the basis of amino acid analysis and the spectrophotometric method, respectively. An amino derivative was prepared by reducing the nitro group with sodium dithionite. The apoadrenodoxin derivatives could be reconstituted to have an iron-sulfur chromophore similar to the native adrenodoxin which contains a 1:1 molar ratio of labile sulfur to iron content and displays absorption peaks at 414 and 450 nm. The enzymatic acitivies of these reconstituted nitro and amino derivatives toward cytochrome c reduction in the presence of adrenodoxin reductase and NADPH were 19 and 7% of native adrenodoxin, respectively. We studied the kinetics of the direct reduction of the reconstituted amino derivative in the presence of NADPH and adrenodoxin reductase under anaerobic conditons. The initial rate of reduction for the amino derivative was 7% of the native adrenodoxin, which is in good agreement with its activity toward cytochrome c reduction. From these results, it is concluded that by modifying the tyrosyl residue at position 82 of the adrenodoxin polypeptide, the electron-transferring activity of the molecule is largely diminished.     

Structural and functional characterization of bovine adrenodoxin reductase by limited proteolysis

Previously, we have proposed that bovine adrenocortical mitochondrial adrenodoxin reductase may possess a domain structure, based upon the generation of two major peptide fragments from limited tryptic proteolysis. In the present study, kinetic characterization of the NADPH-dependent ferricyanide reductase activity of the partially proteolyzed enzyme demonstrates that Km(NADPH) increases (from 1.2 μM to 2.7 μM), whereas 1 V_max remains unaltered at 2100 min⁻¹ The two proteolytic fragments have been purified to homogeneity by reverse-phase HPLC, and amino acid sequence analysis unambiguously demonstrates that the 30.6 kDa fragment corresponds to the amino terminal portion of the intact protein, whereas the 22.8 kDa fragment is derived from the carboxyl terminus of the reductase. Trypsin cleavage occurs at either Arg-264 or Arg-265. Covalent crosslinking experiments using a water-soluble carbodiimide show that adrenodoxin crosslinks exclusively to the 30.6 kDa fragment, thus implicating the N-terminal region of adrenodoxin reductase in binding to the iron-sulfur protein. Our inability to detect covalent carbohydrate on either intact or proteolyzed adrenodoxin reductase prompted a re-examination of the previously reported requirement of an oligosaccharide moiety for efficient electron transfer from the reductase to adrenodoxin. Treatment of adrenodoxin reductase with a highly purified preparation of neuraminidase demonstrates that neither the adrenodoxin-independent ferric yanide reductase activity nor the adrenodoxin-dependent cytochrome c reductase activity of the enzyme is affected by neuraminidase treatment.     

Conformational change of adrenodoxin induced by reduction of iron-sulfur cluster. Proton nuclear magnetic resonance study.

Bovine adrenodoxin in the reduced form has been measured by one- and two-dimensional 1H NMR spectroscopy. By comparing the spectrum of reduced adrenodoxin with that of the oxidized protein, resonances have been assigned for the aromatic residues. The spin-lattice relaxation time for the resonances due to histidine residues was found to depend on the reduction state of adrenodoxin. The distance from the paramagnetic center is calculated by using the Solomone-Bloembergen equation. The resonances from Tyr-82 and Ala-81 show large chemical shift changes upon reduction of adrenodoxin. The conformational change of adrenodoxin manifested by chemical shift difference between reduced and oxidized forms is found in the sequence around Tyr-82 and Ala-81. Modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at Glu-74, Asp-79, and Asp-86 inhibited the interaction with both adrenodoxin reductase and cytochrome P-450scc (Lambeth, D. J., Geren, L. M., and Millett, F. (1984) J. Biol. Chem. 259, 10025-10029; Geren, L. M., O'Brien, P., Stonehuerner, J., and Millett, F. (1984) J. Biol. Chem. 259, 2155-2160). Thus, the sequence of these amino acids was assigned to the interaction site with the redox partners. The present 1H NMR investigation of adrenodoxin demonstrates that a conformational change upon reduction of the iron-sulfur cluster occurs in the sequence of negatively charged amino acids that is a putative site for interaction with redox partners. This could offer the structural basis of the electron transfer mechanism in which adrenodoxin functions as a mobile electron carrier.     

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.     

Molecular dynamics simulation of truncated bovine adrenodoxin

The 128 amino acid long soluble protein adrenodoxin (Adx) is a typical member of the ferredoxin protein family that are electron carrier proteins with an iron-sulfur cofactor. Adx carries electrons from adrenodoxin reductase (AdR) to cytochrome P450s. Its binding modes to these proteins were previously characterized by site-directed mutagenesis, by X-ray crystallography for the complex Adx:AdR, and by NMR. However, no clear evidence has been provided for the driving force that promotes Adx detachment from AdR upon reduction. Here, we characterized the conformational dynamics of unbound Adx in the oxidized and reduced forms using 2-20 ns long molecular dynamics simulations. The most noticeable difference between both forms is the enhanced flexibility of the loop (47-51) surrounding the iron-sulfur cluster in the reduced form. Together with several structural displacements at the binding interface, this increased flexibility may be the key factor promoting unbinding of reduced Adx from AdR. This points to an intrinsic property of reduced Adx that drives dissociation.     

Ionic effects on adrenal steroidogenic electron transport. The role of adrenodoxin as an electron shuttle.

We have shown (Seybert, D., Lambeth, D., and Kamin, H. (1978), J. Biol. Chem. 253, 8355-8358) that, whereas the 1:1 complex between adrenodoxin reductase and adrenodoxin is the active species for cytochrome c reduction, the complex is not sufficient to allow cytochrome P-45011 beta-mediated hydroxylations;adrenodoxin in excess of reductase is required. In the present studies, reduction by NADPH of excess adrenodoxin is shown to occur at a rate sufficient to support both cytochrome P-450 11 beta-mediated hydroxylation of deoxycorticosterone, and cytochrome P-450sec-mediated side chain cleavage of cholesterol. Oxidation-reduction potential and ion effect studies indicate that the mechanism of steroidogenic electron transport involves an adrenodoxin electron "shuttle" rather than a macromolecular complex of reductase, adrenodoxin, and cytochrome. The oxidation-reduction potential of adrenodoxin is shifted about -100 mV when bound to reductase, and reduction of the iron-sulfur protein thus promotes dissociation of the complex. The rate of adrenodoxin reduction is first stimulated, then inhibited by increasing salt; the effect is ion-specific, with Ca2+ approximately Mg2+ greater than Na+ greater than NH/+. Similar ion-specific rate effects are observed for both of the cytochrome P-450-mediated hydroxylations, indicating that the same reduction mechanism is required for these reactions. Increasing salt concentrations caused dissociation of the complex; dissociation of the form of the complex containing reduced adrenodoxin occurred at lower salt concentrations than that containing oxidized adrenodoxin. The order of effectiveness of ions in causing dissociation is the same as the order for stimulation of adrenodoxin reduction, suggesting a dissociation step in the mechanism. This proposed model, together with dissociation constants for the form of the complex containing either oxidized or reduced adrenodoxin, allows accurate prediction of the salt rate effects curve. For all ions, an activity maximum is seen at the ion concentration which produces the largest molar difference between associated-oxidized and dissociated-reduced states, and the model predicts the positions of the maxima for adrenodoxin reduction, 11 beta-hydroxylation, and side chain cleavage. Thus reduction-induced dissociation of adrenodoxin from adrenodoxin reductase appears to be a required step in steroidogenic electron transport by this system, and a role for adrenodoxin as a mobile electron shuttle is proposed.     

Purification and catalytic properties of a cross-linked complex between adrenodoxin reductase and adrenodoxin

A stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c.     

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the "tac" promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.     

Identification of free and [Fe2S2]-bound cysteine residues of adrenodoxin

Bovine adrenodoxin was labeled with 5-iodoacetamidofluorescein to determine which of the five cysteines is free and which participate in iron coordination. Native protein was labeled at two stoichiometries, 0.15:1 and 1:1, both of which produced a single fluorescent product. Labeled tryptic peptides were isolated from both preparations and identified as residues 90-98 with 5-acetamidofluorescein cysteine at residue 95. From the preparation labeled at 0.15:1 stoichiometry, the fraction of tryptic peptide containing nonlabeled cysteines 92 and 95 was isolated and identified; this peptide was shown to be absent in the sample labeled at 1:1 stoichiometry. 5-Acetamidofluorescein-labeled adrenodoxin supported electron transport with adrenodoxin reductase and cytochromes P-450sec and P-45011 beta, demonstrating that labeling occurred without disruption of the iron-sulfur center. These results identify cysteine 95 as the most reactive and single free thiol in native adrenodoxin and imply the role of cysteine residues 46 [corrected], 52, 55, and 92 in iron-sulfur coordination.     

Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.     

A negative cooperativity between NADPH and adrenodoxin on binding to NADPH:adrenodoxin reductase

Adrenodoxin stimulated the oxidation of NADPH by 1,4-benzoquinone, catalyzed by NADPH:adrenodoxin reductase. It prevented the enzyme inhibition by NADPH and formed an additional pathway of benzoquinone reduction presumably via reduced adrenodoxin. In the presence of 100-400 microM NADP+, which increased the Km of NADPH, adrenodoxin acted as a partial competitive inhibitor for NADPH decreasing its TN/Km by a limiting factor of 3. Ki of adrenodoxin decreased on the NADP+ concentration decrease and was estimated to be about 10(-8) M in the absence of NADP+.     

The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex. The implication in ACTH function.

Binary and ternary complexes of bovine adrenocortical mitochondrial cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex are formed in the presence of cholesterol and Emulgen 913. Both cholesterol and Emulgen 913 are required for the binding of cytochrome P-450scc with adrenodoxin. Since phospholipids are able to replace Emulgen 913 in this reaction, in vivo phospholipids of the mitochondrial inner membrane appear to play the function of the detergent. The dissociation constants of the cytochrome.adrenodoxin complex are 0.3 to 0.4 microM at 130 microM dimyristoylphosphatidylcholine and 0.9 microM at 120 microM Emulgen 913, whereas the dissociation constant for the ternary complex of cytochrome P-450scc with adrenodoxin reductase and adrenodoxin is 4.0 microM at 150 microM Emulgen 913. The stoichiometry of binary and ternary complexes reveals the 1:1 and 1:1:1 molar ratios, respectively, judging from chemical analyses after the fractionation of the complexes by gel filtration. Emulgen 913, Tween 20, ethylene glycol, myristoyllysophosphatidylcholine, dimyristoylphosphatidylcholine, and phosphatidylethanolamine show the enhanced activity of cholesterol side chain cleavage reaction with cytochrome P-450scc, adrenodoxin, adrenodoxin reductase, and NADPH. These results, in conjunction with earlier experiments, lead us to the proposal on the structure of the hydroxylase complex in the membrane and to the hypothesis on the regulation of the enzymatic activity by the availability of substrate cholesterol to the cytochrome. Hence, we propose a mobile P-450scc hypothesis for the response of the mitochondrion to adrenocorticotropic hormone stimuli.     

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.     

Structural organization of adrenodoxin using limited proteolysis

The microheterogeneity of adrenodoxin preparation was established by endogenous proteolysis. The controlled limited trypsinolysis and endogenous proteolysis result in modification of the COOH-terminus of the polypeptide chain with a formation of a protein with Mr = 10 000. The interaction of this protein and of the native protein with cholesterol-specific cytochrome P-450 and adrenodoxin reductase occurs in a similar way.     

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.     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

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...     

Stimulation of the NADPH:adrenodoxin reductase diaphorase reaction by adrenodoxin

The diaphorase activity of NADPH: adrenodoxin reductase (EC 1.18.1.2) is stimulated by adrenodoxin. The latter prevents the reductase inhibition by NADPH; the Line-weaver-Burk plots are characterized by a biphasic dependence of the reaction rate on the oxidizer concentration. At pH 7.0 the maximal rate of the first phase is 20s-1; that for the second phase at saturating concentrations of adrenodoxin is 5 s-1. Since the second phase rate is equal to that of the adrenodoxin-linked cytochrome c reduction by reductase it is concluded that this phase reflects the reduction of the oxidizers via reduced adrenodoxin. Quinones are reduced by adrenodoxin in an one-electron way; the logarithms of their rate constants depend hyperbolically on their single-electron reduction potentials (E7(1]. The oxidizers interact with a negatively charged domain of adrenodoxin. The depth of the adrenodoxin active center calculated from the Fe(EDTA)- reduction data is 5.9 A.     

A rubredoxin-like mononuclear FeS4 derivative of adrenal iron-sulfur protein (adrenodoxin)

A rubredoxin-like mononuclear iron-sulfur derivative of adrenodoxin was prepared from the apoprotein and FeCl₃ in the presence of dithiothreitol. The mononuclear compound displayed optical absorption maxima at 276, 350, and 500 nm, and exhibited electron paramagnetic resonance absorption at g = 4.27 with a shoulder at g = 4.28, which can be ascribed to high spin ferric ion. From p-chloromercuriphenyl sulfonate titration experiments the iron atom appears to contain approximately one g atom of iron per mole of protein. This rubredoxin-like derivative was very unstable at 22° (the half-life was approximately 10 minutes), whereas the native 2 Fe2S1 protein is known to be quite stable. This instability is believed to be intrinsic to the polypeptide sequence of adrenodoxin.     

The role of tryptophanyl residues in electron transfer from NADPH--adrenodoxin reductase to adrenodoxin

Chemical modification of tryptophanyl residues of NADPH - adrenodoxin reductase by N - bromosuccinimide and trichloroethanol prevents the interaction of the enzyme with adrenodoxin. The modification does not touch other amino acid residues besides tryptophan (tyrosine, lysine and cysteine) or disturb the structure of protein. The presence of adrenodoxin suppresses the modification. The data obtained indicate the participation of adrenodoxin reductase tryptophan residues in the interaction with adrenodoxin.