Topological studies of the steroid hydroxylase complexes in bovine adrenocortical mitochondria.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria was studied by using nonpenetrating artificial electron acceptors and the impermeable protein reagent diazobenzenesulfonate. Inhibition of steroid hydroxylase activity by ferricyanide and dichlorophenolindophenol sulfonate was only observed in mitochondria which had been damaged by various techniques. Intact mitochondria were not inhibited by these reagents. The reaction was monitored by oxygen uptake due to hydroxylation of deoxycorticosterone, as well as P-450 reduction and corticosterone formation. The results obtained were similar regardless of how the activity was measured. Labeling of the mitochondria with the nonpenetrating protein reagent diazobenzenesulfonate also inhibited P-450 reduction and corticosterone formation in mitochondria which had been damaged prior to addition of this reagent. Intact mitochondria which were labeled with this reagent showed very little inhibition of both activities. These results strongly suggest that all protein components of the steroid 11beta-hydroxylase system are located on the matrix side of the mitochondrial inner membrane. The inability of ferricyanide, dichlorophenolindophenol sulfonate, and diazobenzenesulfonate to inhibit the malate-dependent reduction of P-450 in intact mitochondria implies that all the P-450-dependent mitochondrial steroid hydroxylase systems are located on the matrix side of the inner mitochondrial membrane.     

Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.     

Organ-specific properties of cytochromes P-450s21 (steroid 21-hydroxylases) of liver and adrenocortical microsomes

Prothrombin, plasminogen, urokinase- and tissue-type plasminogen activators contain homologous structures known as kringles . The kringles correspond to autonomous structural and folding domains which mediate the binding of these multidomain proteins to other proteins. During evolution the different kringles retained the same gross architecture, the kringle -fold, yet diverged to bind different proteins. We show that the amino acid sequences of the type II structures of the gelatin-binding region of fibronectin are homologous with those of the protease- kringles . Prediction of secondary structures revealed a remarkable agreement in the positions of predicted beta-sheets, suggesting that the folding of kringles and type II structures may also be similar. As a corollary of this finding, the disulphide-bridge pattern of type II structures is shown to be homologous to that in kringles . It is noteworthy that protease- kringles and fibronectin type II structures have similar functions inasmuch as they mediate the binding of multidomain proteins to other proteins. It is proposed that the kringles of proteases and type II structures of fibronectin evolved from a common ancestral protein binding module.     

Inhibition of steroid C-21 hydroxylase by ascorbate: Alterations of microsomal lipids in beef adrenal cortex

The steroid C-21 hydroxylase system of beef adrenal cortex microsomes is inhibited by low concentrations of ascorbic acid with concomitant production of a thiobarbituric acid (TBA)-reacting chromogen. Antioxidants such as cobalt, manganese, epinephrine or tocopherol prevent production of this chromogen and decrease the effect of ascorbic acid on hydroxylase activity. The production of the TBA-reacting material is associated with a diminution of arachidonic acid in microsomal phospholipid.     

The activation of microsomal steroid 21-hydroxylase by cytosol from the cortex of bovine adrenals.

At low microsome concentrations, the addition of cytosol from bovine adrenal cortex markedly accelerates the rate of hydroxylation of 17-hydroxyprogesterone at C-21. The detection of this effect was made possible by the development of a new, rapid, and sensitive procedure for the measurement of the initial rate kinetics of steroid 21-hydroxylase. This procedure is based on the fact that the reactant, 17-hydroxyprogesterone, possesses solvent partition properties which are different from those of the product, cortexolone. The specificity of the assay was confirmed by the isolation of only one product which was identified as cortexolone by radiochemical techniques. This assay procedure has great sensitivity and makes possible the accurate determination of the Michaelis constant at low enzyme concentrations. The Km for 17-hydroxyprogesterone with saturating amounts of TPNH was found to be 0.3 micronM. At the low microsome concentrations permitted by this assay, the addition of cytosol has a profound effect upon the rate of hydroxylation. The rate is markedly accelerated although the Km for the substrate is not altered. Neither the substrate nor the carbon monoxide-induced difference spectra are changed by the addition of cytosol, suggesting that activation by cytosol dose not affect the catalytic unit of the 21-hydroxylase complex.     

Effect of spin state on reduction of cytochrome P-450 (P-450C21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450C21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17 alpha-hydroxyprogesterone-bound P-450C21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25 degrees C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450C21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25 degrees C. The steady-state results showed that the rate of P-450C21 reduction was not affected by the high spin content of substrate-bound P-450C21 but was very slow without a steroid substrate. Biphasic reduction of P450C21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17 alpha-hydroxyprogesterone-bound and progesterone-bound P-450C21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450C21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450C21.     

Kinetics of cytochrome P-450 reduction: studies in bovine adrenocortical microsomes

The results of the present study indicate first that in the microsomal preparation, the components of the P-450 reduction system are heterogeneously distributed, comprising dissociable and nondissociable parts. Second, the P-450 reduction curve can be adequately described by a sum of two exponential functions, indicating two concurrent first-order reactions. Third, the two phases can be altered independently. The addition of the substrate increased the extent of the fast phase while it had little or no effect on that of the slow phase. Changes in the interaction of the dissociable and nondissociable components affected the extent of the slow phase while they were without effect on that of the fast phase. Experiments with different steroids indicated that the independence of the two phases is not due to functionally different P-450's and that the cytochrome reduced in both phases is essentially P-450C-21. The results are interpreted as follows: Transformation of P-450 from the low- to the high-spin state controls the total P-450 reduced. The rate and the biphasicity of the reduction are functions of the interaction of P-450 and the reductase.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

Multiple regulatory elements determine adrenocortical expression of steroid 21-hydroxylase

Steroid 21-hydroxylase (21-OHase) is specifically expressed at high levels in the adrenal cortex, where it is required for the synthesis of mineralocorticoids and glucocorticoids. In this study, we have investigated the regulatory elements in the 21-OHase promoter region which contribute to the expression of this gene in Y1 adrenocortical cells. Eight potential regulatory elements in the 5'-flanking region of the 21-OHase gene were identified by DNase I footprinting and gel mobility shift experiments. Some of these footprints were produced by nuclear extracts from many cell lines, whereas other interactions were seen only when using nuclear extracts from Y1 adrenocortical and MA-10 Leydig tumor cells. Mutation of most of the elements markedly decreased the expression of a 21-OHase gene transfected into Y1 cells, thus documenting their functional importance for expression. Moreover, oligonucleotides containing the sequences of two related elements at -65 and -210, which share the heptamer AGGTCAG, increased the activity of a heterologous promoter in a Y1 cell-specific manner. Collectively, these results demonstrate that expression of 21-OHase in Y1 adrenocortical cells requires interactions among multiple cis-acting elements and regulatory proteins.     

Isolation from bovine liver mitochondria of a soluble ferredoxin active in a reconstituted steroid hydroxylation reaction.

An iron-sulfur protein has been isolated from bovine liver mitochondria and purified 140-fold on DEAE-cellulose and Sephadex G-100. During the isolation the protein was detected by its NADPH-cytochrome $\text{c}$ reductase activity in the presence of adrenal NADPH-ferredoxin reductase. The molecular weight of the protein (12,400), the optical spectrum (peaks at 414 nm and 455 nm which disappear upon reduction), and the EPR spectrum (g_x = g_y = 1.935 and g_z = 2.02) were typical for a ferredoxin. In the presence of soluble adrenal cytochrome P₄₅₀, ferredoxin reductase and NADPH, this protein could support the formation of pregnenolone from cholesterol. Under similar conditions, but in the presence of a cytochrome P₄₅₀ solubilized from rat liver mitochondria, cholesterol was transformed into a more polar compound tentatively identified as 26-hydroxycholesterol.     

An intramembranous reductant which participates in the proline hydroxylation reaction with intracisternal prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells

We previously have described a substance present in crude sonicates of L-929 cells which replaced ascorbate in vitro as a reductant for prolyl hydroxylase (B. Peterkofsky, D. Kalwinksy and R. Assad, 1980, Arch. Biochem. Biophys.199, 362–373). In the present study we found that almost 90% of the substance was particulate after differential centrifugation of stationary phase L-929 cell homogenates. The substance was not localized in nuclei or mitochondria and was found in the same fractions as microsomes, but these fractions also contained lysosomes and cell membranes. The reductant could not be solubilized from particles by Brij-35, indicating that it is an intrinsic component of a membrane rather than intracisternally located. The intramembranous cofactor, in the absence of ascorbate, participated in the in vitro hydroxylation of [4-³H]proline in radio-actively labeled, intracisternal unhydroxylated procollagen in isolated microsomes which also contained prolyl hydroxylase. Hydroxylation was determined by measuring tritiated water formed from release of the 4-trans tritium atom. Since it is unlikely that such participation could occur if the cofactor were located within the membrane of another subcellular organelle, we have concluded that it is in the same particle as prolyl hydroxylase and unhydroxylated procollagen, that is, the microsome. With the endogenous reductant the reaction was slower than with saturating ascorbate and was increased by NADH. Maximum hydroxylation with the endogenous reductant was close to that which could be achieved with ascorbate. These results provide strong evidence that the endogenous reductant alone can account for the phenomenon of ascorbate-independent proline hydroxylation in L-929 cells. As in the case of ascorbate, the microsomal reductant functioned only in the presence of α-ketoglutarate and Fe²⁺ and served as reductant for lysyl hydroxylase. It also was detected in the particulate fraction of virally transformed BALB 3T3 cells and in purified microsomes from bones of intact chick embryos. Since ascorbate could be taken up and concentrated in bone microsomes, it is unlikely that the endogenous reductant serves as an intermediary between ascorbate and intracisternal prolyl hydroxylase.     

Fluoride elimination from substrates in hydroxylation reactions catalyzed by p-hydroxybenzoate hydroxylase

Several fluorinated derivatives of p-hydroxybenzoate were synthesized and examined as substrates in the reaction catalyzed by p-hydroxybenzoate hydroxylase. All the derivatives tested served as substrates, undergoing tightly coupled hydroxylation by molecular oxygen. Hydroxylation of the difluoro and tetrafluoro derivatives liberated stoichiometric amounts of fluoride. Little or no fluoride was released with monofluoro substrates. The defluorination caused higher consumption of NADPH with an overall NADPH to oxygen ratio of 2, in contrast to the ratio of 1 with the physiological substrate and with the monofluoro derivatives. Evidence was obtained strongly suggestive of a quinonoid species as the primary product formed upon oxygenative defluorination. The additional equivalent of NADPH consumed upon fluoride elimination is presumably used in a nonenzymatic reaction with the quinonoid intermediate, resulting in the observed dihydroxy product. Stopped flow studies of the reductive and oxidative half-reactions with tetrafluoro-p hydroxybenzoate substrate were examined. The oxygen half-reaction was analogous to that with p-hydroxybenzoate involving two transient oxygenated flavin intermediates. The decay of the first intermediate, a C(4a)-peroxyflavin, results in rupture of the oxygen-oxygen bond and is rate-determining in overall catalysis. This is in contrast to the reaction with the normal substrate, presumably due to a deactivating effect of the fluorine substituents. The above results are consistent with an oxenoid mechanism of oxygen attack.