The pH dependence of lipid peroxidation using water-soluble azo initiators.

We report here the pH dependence of the rate of lipid peroxidation of methyl linoleate/Triton mixed micelles using a series of water-soluble azo initiators. The cationic initiators 2,2'-azobis(2-amidinopropane) (ABAP) and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (ABIP) exhibit similar behavior, in which increased pH results in dramatically enhanced rates of peroxidation. Rate data for ABAP and ABIP were fitted to a single proton equilibrium, which yielded apparent kinetic pKa values for the rate of approximately 7 and 6, respectively. The azo initiator 4,4'-azobis(4-cyanopentanoic acid) (ABCPA), which yields a negatively charged radical upon thermolysis at neutral pH, was also studied. In contrast to the effects observed with ABAP and ABIP, peroxidation rates with ABCPA exhibit an inverse pH dependence, in which the rates of peroxidation increase with decreasing pH, with an apparent pKa of approximately 5. By comparison, methyl linoleate oxidation rates with 2,2'-azobis (2-cyanopropane) (ABCP) display minimal changes over the pH range 5 to 7.5. Two alternatives can be advanced to account for this behavior, including either a buffer effect which is specific to the cationic initiators or an altered amidinium pKa (approximately 6 to 7) in either the initial carbon-centered radical or the subsequent peroxyl radical generated upon thermolysis of ABAP or ABIP. In the latter case, the kinetic pH dependence could thus reflect an enhanced competence of neutral radicals over charged radicals to partition into the micelles and initiate peroxidation.

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 microM to 2.7 microM), whereas Vmax 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 ferricyanide reductase activity nor the adrenodoxin-dependent cytochrome c reductase activity of the enzyme is affected by neuraminidase treatment.

Modified acetylenic steroids as potent mechanism-based inhibitors of cytochrome P-450SCC.

Synthesized 20-(4-tetrahydropyranyl-1-butynyloxy)-5-pregnen-3 alpha,20 beta- diol [steroid I] and 20-(3-tetrahydropyranyl-1-propargyloxy)-5-pregnen- 3 alpha,20 beta-diol [steroid III] have been found to inactivate purified adrenocortical cytochrome P-450SCC. When incubated with the enzyme under turnover conditions, steroid I inactivated cytochrome P-450SCC by about 85% in 40 min. This is in contrast to the free triol analog, steroid II which inactivated the enzyme by only 45% within the same incubation period. A comparison of steroid III with its free triol analog, steroid IV, also showed that the diol is a more effective inactivator of the enzyme than the triol. The partition ratio was calculated by two different methods. Each of the steroids I-IV bound to the enzyme with spectrophotometric dissociation constant (Ks) in the micromolar range, producing Type II low spin spectra changes during titration of the enzyme. In addition, it was found that the binding of each of the compounds to the enzyme occurred without inactivation of the enzyme and that the inactivation under turnover condition, is not as a result of conversion to the denatured P-420 species. This demonstrated that steroids I and III could correctly be designated as mechanism-based (suicide) inhibitors. The kinetic studies demonstrated that steroids with the tetrahydropyranyl substituent are more potent inhibitors of cytochrome P-450SCC as shown by an initial turnover rate of 0.06 min-1, an inactivation rate constant of 0.05 min-1, and a partition ratio of about 1.0 for steroid I. Based on our finding, possible mechanisms of inactivation of cytochrome P-450SCC by these acetylenic steroids are proposed.

Lipid regulation of bovine cytochrome P450(11) beta activity.

Purified bovine adrenocortical cytochrome P450(11) beta has been reconstituted into phospholipid vesicles using a detergent dialysis procedure. Using this reconstituted system, we have examined the effect of changes in the fatty acyl substituents of the lipids on the catalytic activity of the enzyme. The studies reported here show that cytochrome P450(11) beta exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450scc. Cytochrome P450(11) beta displays maximal activity in lipid vesicles composed of saturated lipids, such as dipalmitoyl and dimyristoyl phosphatidylcholines, with turnover numbers ranging from 35 to 60 min-1. Incremental increases of phospholipids such as diphytanoyl and dioleoyl phosphatidylcholines result in a progressive inhibition of 11 beta hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in Vmax accompanied by modest changes in Km for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450scc, is a potent inhibitor of the 11 beta hydroxylase activity of cytochrome P450(11) beta, with half maximum inhibition observed in vesicles containing 4-5 mol% diphosphatidyl glycerol. Kinetic analysis demonstrates that this inhibition by diphosphatidyl glycerol is reflected in both a decrease in Vmax and relatively large increases (up to sevenfold) in Km for the steroid substrate. These effects on the 11 beta hydroxylase activity may have important implications for the in vivo regulation of not only the 11 beta hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.

Limited proteolysis of bovine adrenodoxin reductase: evidence for a domain structure.

Treatment of bovine adrenodoxin reductase with trypsin under conditions of limited proteolysis yields two major fragments of apparent molecular weights 30,500 and 20,200. The fragments, which have been partially purified by affinity chromatography to remove most of the intact adrenodoxin reductase, retain adrenodoxin-dependent NADPH cytochrome c reductase activity. Kinetic analyses yield Vmax and Km (adrenodoxin) values of 485 min-1 and 0.96 microM, respectively, at an ionic strength of 0.13 M in comparison to 1059 min-1 and 0.40 microM, respectively, for intact adrenodoxin reductase under the same conditions.

Steroidogenic electron transport in adrenal cortex mitochondria.

The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxin-adrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450scc to which this phospholipid binds.

Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome.

Cytochrome P-450scc from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's Km for cholesterol, and not due to Vmax effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated versus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-45scc is embedded.

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.

Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.

A structural model for the kinetic behavior of hemoglobin.

The tertiary structures of all liganded hemoglobins in the R state differ in detail. Steric hindrance arising from nonbonded ligand-globin interactions affects the binding of ligands such as CO and cyanide which preferentially form linear axial complexes to heme; these ligands bind in a strained off-axis configuration. Ligands such as O2 and NO, which preferentially form bent complexes, encounter less steric hindrance and can bind in their (preferred) unstrained configuration. Linear complexes distort the ligand pockets in the R state (and by inference, in the T state) more than bent complexes. These structural differences between linear and bent complexes are reflected in the kinetic behavior of hemoglobin. Structural interpretation of this kinetic behavior indicates that the relative contributions of nonbonded ligand-globin interactions and nonbonded heme interactions to transition state free energies differ for linear and bent ligands. The relative contributions of these interactions to the free energy of cooperativity may also differ for linear and bent ligands. Thus the detailed molecular mechanism by which the affinity of heme is regulated differs for different ligands.

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.

The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation.

We have utilized 11beta-hydroxylase activity and visible absorption spectrophotometry to detect possible complex formation among adrenodoxin reductase, adrenodoxin, and cytochrome P-450(11)beta. At low ionic strength, a 1:1 complex between adrenodoxin reductase and adrenodoxin occurs but does not support maximal rates of 11beta hydroxylation; at least 1 additional molecule of adrenodoxin in excess of the 1:1 complex is required for full hydroxylase activity. Spectrophotometric titration of a mixture of adrenodoxin reductase and cytochrome P-450(11)beta with adrenodoxin indicates sequential formation of 1:1 complexes between adrenodoxin reductase and adrenodoxin and then between a second adrenodoxin and cytochrome P-450(11beta; the adrenodoxin-cytochrome P-450(11)beta complex is only detectable when the concentration of adrenodoxin exceeds that of adrenodoxin reductase.

Ligand binding properties of horse hemoglobins containing deutero- and mesoheme.

The reactions of horse globin reconstituted with proto-, deutero-, and mesoheme have been examined by equilibrium and kinetic methods. In virtually all reactions studied, mesohemoglobin displays the more extreme functional behavior, whereas deuterohemoglobin exhibits behavior which is either very similar to native hemoglobin or intermediate between the two. Our kinetic and equilibrium results indicate that the primary effect of heme modification on the functional properties of hemoglobin is to alter the intrinsic reactivities of the deoxy and liganded conformations. Heme modification does not, however, result in substantial alterations in the conformational equilibrium between the two states. Simple inductive electronic effects of the 2- and 4-substituents of the heme moiety in deutero- and mesohemoglobin are apparently not sufficient to explain the observed equilibrium and kinetic properties completely, which indicates that steric effects of these substituents may also play a role in determining the functional behavior of the hemoglobin molecule.