The study of the active site of cytochrome P-450 LM2 using the chemical modification of tyrosine residues by tetranitromethane

Selective chemical modification of the hemoprotein by tetranitromethane was used in order to elucidate the functional role of tyrosine residues in the cytochrome P-450 LM2 molecule. It was shown that the degree of cytochrome P-450 LM2 modification can be determined, using the second derivative of the UV absorption spectra. Modification of one tyrosine residue resulted in the inactivation of cytochrome P-450 LM2. Nitration of the cytochrome was accompanied by changes in the spectral properties of the hemoprotein with the formation of spectra typical of hyperporphyrin structures, thus suggesting the involvement of tyrosine residues in the formation of the active center of cytochrome P-450 LM2.     

Chemical modification of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria with diethylpyrocarbonate

Chemical modification of adrenocortical cytochrome P-450scc with diethyl pyrocarbonate has been carried out. The histidine residues and the protein amino groups were shown to undergo modification. Carbethoxylation was accompanied by the hemoprotein inactivation and the loss of enzymatic activity. Neither of the high spin effectors (i.e., substrate and adrenodoxin) protected cytochrome P-450scc either from inactivation or from the loss of enzymatic activity. The data obtained are discussed in terms of the functional role of histidine residues in the cytochrome P-450scc molecule.     

Chemical modification of cytochrome P-450 LM2. Characterization of tyrosine as axial heme iron ligand trans to thiolate

Phenobarbital-inducible isozyme cytochrome P-450 LM2 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes has been modified with N-acetylimidazole and tetranitromethane. Up to four tyrosine residues of cytochrome P-450 LM2 are accessible to O-acetylation and to nitration. N-Demethylase activity, spectral dissociation constants and substrate binding kinetics of differently acetylated enzyme indicate the existence of two groups of accessible tyrosines also differing in their reactivity towards N-acetylimidazole. The fast-reacting tyrosine residue representing the first group is involved in the binding of the type II substrate aniline and appears to be located near the heme as shown by the protecting effect of the inhibitor metyrapone against modification, but obviously is not necessary for N-demethylation. Acetylation of one further tyrosine residue, however, caused an almost complete inhibition of the enzyme, indicating its involvement in the catalytic mechanism at the active center. Nitration of two tyrosine residues inactivates to about 20%. Obviously the third and fourth tyrosine residue are without functional importance. The experiments evidencing two functionally linked tyrosines are in line with HPLC analyses of tryptic peptides of cytochrome P-450 LM2 nitrated in the presence of metyrapone which gave evidence for the location of two distinct tyrosine residues in the active center. Nitration of tyrosine residues results in the partial formation of a hyperporphyrin spectrum of cytochrome P-450 LM2. Its appearance is prevented in the presence of metyrapone and can be reversed by reduction of the nitrotyrosinate .     

Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.     

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

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

Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue

It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.     

Chemical modification of cysteine residues of cholesterol-hydroxylating cytochrome P-450. Identification of the cysteine residue participating in the formation of a proximal ligand

A chemical modification of cysteine residues in mitochondrial cytochrome P-450scc from adrenal cortex has been carried out. Cysteine residues in this hemoprotein were shown to form two pools: one available to the chemical modification, which does not affect spectral and functional properties of the cytochrome and another accessible for the modification only after the protein inactivation and the heme removal. The proximal ligand in the polypeptide chain of cytochrome P-450scc was localized. Cys422 was shown to be involved in the heme coordination and Cys264 was found to be exposed and accessible to sulfhydryl reagents. The data obtained are discussed in terms of the functional role of cysteine residues in monooxygenase catalysis.     

Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.     

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.     

Chemical modification of cholesterol-hydroxylating cytochrome P450 from bovine adrenal cortex mitochondria by acetylimidazole

The state and role of tyrosine residues in adrenocortical cytochrome P-450scc was investigated. Spectrophotometric titration experiments showed the existence of two types of tyrosine residues in the hemoprotein molecule, i.e., exposed (pK 9.25) and buried (pK 10.75) ones. Chemical modification of the exposed tyrosine residues with N-acetylimidazole was carried out. The data obtained indicate the involvement of these residues in the interaction with adrenodoxin.     

Conformational change of the heme moiety of the ferrous cytochrome P-450scc-phenyl isocyanide complex upon binding of reduced adrenodoxin

Reduction of cytochrome P-450scc(SF) (SF, substrate free) purified from bovine adrenocortical mitochondria with sodium dithionite (Na2S2O4) or with beta-NADPH mediated by catalytic amounts of adrenodoxin and adrenodoxin reductase in the presence of phenyl isocyanide produced a ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex with Soret absorbance maximum at 455 nm having a shoulder at 425 nm. On the other hand, when a preformed cytochrome P-450scc(SF)-adrenodoxin complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum showed drastic changes, i.e., an increase in intensity at 425 nm and a concomitant decrease in intensity at 455 nm. Similar spectral changes could be produced by addition of the same amount of reduced adrenodoxin afterward to the ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex. Titration experiments with adrenodoxin showed that (1) a 1:1 stoichiometric saturation of the spectral change was obtained for both the absorbance increase at 425 nm and the absorbance decrease at 455 nm, (2) there was no spectral change in the presence of 0.35 M NaCl, and (3) there was no spectral change for cytochrome P-450scc(SF) whose Lys residue(s) essential to the interaction with adrenodoxin had been covalently modified with PLP. These results suggest that ternary complex formation of ferrous cytochrome P-450scc(SF)-phenyl isocyanide with reduced adrenodoxin caused a conformational change around the ferrous heme moiety. By analysis of temperature and pH dependencies of the spectral change of the ternary complex, it was suggested that this conformational change may reflect the essential step for electron transfer from reduced adrenodoxin to the ferrous-dioxygen complex of cytochrome P-450scc.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Study of the role of lysine residues of the cholesterol hydroxylating cytochrome P-450 by a method of chemical modification

Chemical modification of cytochrome P-450scc by lysine-specific reagents has been performed. Modification of the hemoprotein was shown to result in the loss of its ability to interact with adrenodoxin. With a view of identifying lysine residues involved in the interaction with adrenodoxin, cytochrome P-450scc was modified by succinic anhydride in the presence of adrenodoxin. After the removal of ferredoxin, the modification was performed with the use of a radioactively labeled reagent. Subsequent hydrolysis of the succinic hemoprotein by chymotrypsin and separation of the peptides obtained by high pressure liquid chromatography resulted in the isolation of seven chymotryptic peptides containing labeled lysine residues. These amino acid sequences were identified. The role of lysine residues of cytochrome P-450scc in complex formation with adrenodoxin is discussed.     

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.     

Adrenodoxin with a COOH-terminal deletion (des 116-128) exhibits enhanced activity

Adrenodoxin, purified from bovine adrenal cortex, was subjected to trypsin cleavage to yield a trypsin-resistant form, designated TT-adrenodoxin. Sequencing with carboxypeptidase Y identified the trypsin cleavage site as Arg-115, while Edman degradation indicated no NH2-terminal cleavage. Native adrenodoxin and TT-adrenodoxin exhibited similar affinity for adrenodoxin reductase as determined in cytochrome c reductase assays. In side chain cleavage assays using cytochrome P-450scc, however, TT-adrenodoxin demonstrated greater activity than adrenodoxin with cholesterol, (22R)-22-hydroxycholesterol, or (20R,22R)-20,22-dihydroxycholesterol as substrate. This enhanced activity is due to increased affinity of TT-adrenodoxin for cytochrome P-450scc; TT-adrenodoxin exhibits a 3.8-fold lower apparent Km for the conversion of cholesterol to pregnenolone. TT-Adrenodoxin was also more effective in coupling with cytochrome P-450(11) beta, exhibiting a 3.5-fold lower apparent Km for the 11 beta-hydroxylation of deoxycorticosterone. In the presence of partially saturating cholesterol, TT-adrenodoxin elicited a type I spectral shift with cytochrome P-450scc similar to that induced by adrenodoxin, and spectral titrations showed that oxidized TT-adrenodoxin exhibited a 1.5-fold higher affinity for cytochrome P-450scc. These results establish that COOH-terminal residues 116-128 are not essential for the electron transfer activity of bovine adrenodoxin, and the differential effects of truncation at Arg-115 on interactions with adrenodoxin reductase and cytochromes P-450 suggest that the residues involved in the interactions are not identical.     

Localization of tetranitromethane-modified tyrosine residues in the polypeptide chain of cholesterol-hydroxylating cytochrome P-450

As a continuation of earlier structure-function relationship studies on cholesterol-hydroxylating cytochrome P-450 from the adrenal cortex mitochondria, the present study deals with the distribution of tetranitromethane-modified tyrosine residues in the hemeprotein polypeptide chain. Amino acid residues Tyr-24, -46, -50, -93, -94, -199, -246 are shown to be modified with tetranitromethane. Tyr-93, -94 are supposedly involved in the active site formation of cytochrome P-450.     

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.     

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria

A homogeneous cytochrome P-450scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregnenolone to be catalyzed by single species of cytochrome P-450scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with Mr 27000 and 22000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, Mr of about 14000. Cytochrome P-450scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.     

Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor

Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.     

Purification and comparative characterization of cytochrome P-450scc from porcine adrenocortical mitochondria.

Cytochrome P-450scc (cholesterol side-chain cleavage enzyme) was purified from porcine adrenocortical mitochondria. 2. The purified cytochrome P-450scc was found to be homogeneous on SDS-polyacrylamide gel electrophoresis. 3. The heme content of the purified enzyme was 20.6 nmol/mg protein. 4. The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system amounted to 7.8 nmol of pregnenolone formed per nmole of P-450 per minute, with cholesterol as a substrate. 5. The amino acid sequence of the amino-terminal region of the cytochrome P-450scc and the amino acid residue at the carboxyl terminal were determined and compared with those of other mammalian cytochromes P-450scc.