Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.     

An NADPH-induced change in lipid bilayer of rat liver microsomes as observed by spin-labeled phosphatidylcholine.

Lipid bilayer of rat liver microsomes was spin-labeled by incubating with liposomes of 1-acyl 2-(12-doxylstearoyl) glycero-3-phosphorylcholine. When NADPH was added to the labeled microsomes, there appeared a rapidly tumbling component of spin label in the EPR spectrum. NADH was less effective than NADPH. The appearance of the sharp signal was prevented under anaerobic conditions or in the presence of either carbon monoxide, phenyl isocyanide or cytochrome $\text{c}$. The appearance of the rapidly tumbling component in the EPR spectrum was found to be due to the release of spin moiety from the membrane into the aqueous phase. That the release was associated with superoxide anion formation or with lipid peroxidation is unlikely, since 1) superoxide dismutase had little effect, 2) addition of either α-tocopherol or EDTA did not inhibit the release. These observations suggest that electron transfer from NADPH to oxygen $\text{via}$ cytochrome P-450 system induces a physical perturbation in the lipid bilayer resulting in the release of its component into the aqueous phase.     

Spin-labeling studies of rat liver NADPH-cytochrome p450 reductase: conformation and function relationship.

ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.     

Detection of conformational changes in Complex III of the respiratory chain by a maleimido spin label

Changes in the conformation of Complex III (CoQH₂-cytochromec reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochromec. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-³H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells.

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

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. F?rster 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.     

Spin label studies on conformational changes of aphohemoglobin due to heme binding.

Human apohemoglobin (globin) was spin-labeled at the beta-93 sulfhydryl groups with 2,2,5,5-tetramethyl-3-aminopyrrolidine-I-oxyl. Spin-labeled globin exhibited an EPR spectra that is less immobilized than that of spin-labeled hemoglobin, indicating the conformational difference in the vicinity of the label between hemoglobin and globin. Spectrophotometric titration of spin-labeled globin with protohemin showed that 1 mol of globin (on the tetramer basis) combines with 4 mol of hemin, producing a holomethemoglobin spectrophotometrically indistinguishable from native methemoglobin. The EPR spectrum was also changed strikingly upon the addition of protohemin. This change, however, was not proportional to the amount of hemin added, but marked changes occurred after 3 to 4 mol of hemin were mixed with 1 mol of spin-labeled globin. The EPR spectrum of spin-labeled hemoglobin thus prepared was identical with that prepared by direct spin labeling to methemoglobin. These results suggest the preferential binding of hemin to alpha-globin chains in the course of heme binding by globin. This assumption was further confirmed by preparing spin-labeled semihemoglobin in which only one kind of chain contained hemin (alpha h betaO SL and alpha O beta h SL). The EPR spectrum of the alpha h beta O SL molecule showed a slightly immobilized EPR spectrum, similar to that of spin-labeled globin mixed with 50% of the stoichiometric amount of hemin. On the other hand, the alpha O beta h SL molecule showed a distinctly different EPR signal from that of globin half-saturated with hemin, and showed an intermediate spectrum between those of beta h SL and alpha h beta h SL. These results indicate that heme binding to globin chains brings about a major conformational change in the protein moiety and that chain-chain association plays a secondary role. We conclude that hemin binds preferentially to alpha-globin chains and that the conformation of globin changes rapidly to that of methemoglobin after all four hemes are attached to globin heme pockets.     

Interaction of spin-labeled nicotinamide adenine dinucleotide phosphate with chicken liver fatty acid synthase

The spatial relationships between the four reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding sites on chicken liver fatty acid synthase were explored with electron paramagnetic resonance (EPR) and spin-labeled analogues of NADP+. The analogues were prepared by reaction of NADP+ with 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid, with 1,1'-carbonyldiimidazole as the coupling reagent. Several esterification products were characterized, and the interaction of the N3' ester of NADP+ with the enzyme was examined in detail. Both 1H13, 14N and 2H13, 15N spin-labels were used: the EPR spectrum was simpler, and the sensitivity greater, for the latter. The spin-labeled NADP+ is a competitive inhibitor of NADPH in fatty acid synthesis, and an EPR titration of the enzyme with the modified NADP+ indicates four identical binding sites per enzyme molecule with a dissociation constant of 124 microM in 0.1 M potassium phosphate and 1 mM ethylenediaminetetraacetic acid (pH 7.0) at 25 degrees C. The EPR spectra indicate the bound spin-label is immobilized relative to the unbound probe. No evidence for electron-electron interactions between bound spin-labels was found with the native enzyme, the enzyme dissociated into monomers, or the enzyme with the enoyl reductase sites blocked by labeling the enzyme with pyridoxal 5'-phosphate. Furthermore, the EPR spectrum of bound ligand was the same in all cases. This indicates that the bound spin-labels are at least 15 A apart, that the environment of the spin-label at all sites is similar, and that the environment is not altered by major structural changes in the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of a lysine residue of adrenodoxin reductase, essential for complex formation with adrenodoxin

The NADPH-cytochrome c reductase activity of NADPH-adrenodoxin reductase from NADPH to cytochrome c via adrenodoxin was inhibited by pyridoxal 5'-phosphate and other reagents that modified the lysine residues. However, the NADPH-ferricyanide reductase activity was not affected. Loss of the cytochrome c reductase activity could be prevented by adrenodoxin, but not by NADP+. One lysine residue of the adrenodoxin reductase could be protected from the modification with pyridoxal 5'-phosphate by complex formation with adrenodoxin. Loss of the NADPH-cytochrome c reductase activity was not due to the conformational change of the modified adrenodoxin reductase, judging from circular dichroism spectrometric studies.     

EPR studies of iso-1-cytochrome c: effect of temperature on two-component spectra of spin label attached to cysteine at positions 102 and 47

Wild-type iso-1-cytochrome c from Saccharomyces cerevisiae containing naturally occurring cysteine at position 102 and mutated protein S47C (derived from the protein in which C102 had been replaced by threonine) were labeled with cysteine-specific methanethiosulfonate spin label. Continuous wave (CW) electron paramagnetic resonance (EPR) was used to examine the effect of temperature on the behavior of the spin label in the oxidized and reduced forms of wild-type cytochrome c and in the oxidized form of the mutated protein. The computer simulations revealed that the CW EPR spectrum for each form of cytochrome c consists of at least two components [a fast (F) and a slow (S) component], which differ in the values of the rotational correlation times tauRparallel (longitudinal rotational correlation time) and tauRperpendicular (transverse rotational correlation time) and that the relative contributions of the F and S components of the spectra change with temperature. In addition, the values of the rotational correlation times (tauRparallel and tauRperpendicular) for the F component appear to change much more dramatically with the temperature than the respective values for the S component. A large difference between the behavior of the oxidized and reduced wild-type spin-labeled cytochromes c indicates that the temperature-induced unfolding of the protein in the region around C102 progresses more rapidly when cytochrome c is in the oxidized form.     

A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.     

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.     

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.     

Comparison of Ca(II), Cd(II), and Mg(II) titrations of tyrosine-99 spin-labeled bovine calmodulin

Bovine calmodulin, spin-labeled at tyrosine-99, has been utilized in electron paramagnetic resonance (EPR) studies to investigate calmodulin interactions with Ca(II), Cd(II), and Mg(II). The addition of either Ca(II) or Cd(II) to apo-calmodulin results in a complex capable of activating target enzymes, such as 3', 5'-cyclic nucleotide phosphodiesterase (J. M. Buccigross, C. L. O'Donnell, and D. J. Nelson, Biochem. J. 235 677 [1986]), while Mg(II) is known to be incapable of activating calmodulin toward any of its target enzymes. Additions of Ca(II) and Cd(II) to spin-labeled apo-calmodulin gave rise to very similar changes in the EPR spectrum of the bound label, consistent with a dramatic decrease in the mobility of the nitroxide spin-label covalently attached to tyrosine-99. Addition of Mg(II) to spin-labeled apo-calmodulin caused no change in the EPR spectrum of the bound label. Thus, the conformational changes induced by Ca(II) and Cd(II) ion binding to calmodulin, which lead to decreased tyrosine-99 spin label mobility, are clearly not occurring when Mg(II) ion binds. These results are consistent with the results of other spectroscopic studies, which indicate that "activating" metal ions, such as Ca(II) and Cd(II), produce calmodulin conformers that are different from those produced by "inactivating" metal ions, such as Mg(II).     

Study of ubiquinone binding in ubiquinol-cytochrome c reductase by spin labeled ubiquinone derivative

A functionally active, spin labeled ubiquinone derivative, 2,3-dimethoxy -5-methyl-6-{10-(2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl-3-carboxy)-decyl}-1,4-benzoquinone, has been synthesized for the study of ubiquinone binding in ubiquinol-cytochrome $\text{c}$ reductase. When this spin labeled ubiquinone derivative interacted with ubiquinone- and phospholipid-depleted reductase, the spin label was totally immobilized. However, when phospholipids were replenished, the spin label showed mobility behaviour similar to that observed in a hydrophobic environment, indicating that the alkyl side chain of ubiquinone is extended into the hydrophobic region of intact reductase and has some degree of mobility.     

The Role of the Fast Motion of the Spin Label in the Interpretation of EPR Spectra for Spin-Labeled Macromolecules

Abstract

The spin label method was used to observe the nature of the fast motions of side chains in protein monocrystals. The EPR spectra of spin-labeled lysozyme monocrystals (with different orientations of the tetragonal protein crystal in relation to the direction of the magnetic field) were interpreted using the method of molecular dynamics (MD). Within the proposed simple model, MD calculations of the spin label motion trajectories are performed in a reasonable real time. The model regards the protein molecule as frozen as a whole and the spin labeled amino acid residue as unfrozen. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was assembled, and the parameters of the force fields were specified for atoms of the protein molecule, including the spin label. The calculations show that the protein environment sterically limits the area of the possible angular reorientations for the NO reporter group of the nitroxide (within the spin label), and this, in turn, affects the shape of the EPR spectrum. However, it turned out that the spread in the positions of the reporter group in the angle space strictly adheres to the Gaussian distribution. Using the coordinates of the spin label atoms obtained by the MD method within a selected time range and considering the distribution of the spin label states over the ensemble of spin-labeled macro- molecules in a crystal, the EPR spectra of spin-labeled lysozyme monocrystals were simulated. The resultant theoretical EPR spectra appeared to be similar to experimental ones.     

Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta.

The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome.     

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...     

Revealing of the spin-spin interaction due to incorporating a spin label in the carbohydrate parts of immuniglobulins M and G

The EPR spectra of the preparations produced by spin labeling of the carbohydrate parts in monoclonal IgM and normal IgG with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl as the spin label indicate the existence of a rapid spin-spin exchange interaction between two spin labels. In the case of spin-labeled IgM, the carrier of such a spectrum is shown to be a glycopeptide noncovalently bound to IgM; it includes two spin labels and may be detached from the macromolecule by a combination of dialysis and gel filtration.