Yoshiyuki Ichikawa

Reconversion of detergent- and sulfhydryl reagent-produced P-420 to P-450 by polyols and glutathione.

Abstract After treatment with detergents or sulfhydryl reagents, the cytochrome pigment P-420 can be converted back to P-450 by treatment with polyols or reduced glutathione. This reconversion of P-420 to P-450 is highly dependent on temperature, pH, concentration of sodium cholate and incubation time, but ionic strength was of little influence. However, after treatment with alcohols or ketones, P-420 could no longer be reconverted to P-450 by polyols and reduced glutathione. The presence of 20% (v/v) glycerol stabilizes P-450 for more than 1 week.

Existence of Two Heme B Centers in Cytochromeb 561 from Bovine Adrenal Chromaffin Vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy

We have established a new purification procedure of cytochrome b 561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochromeb 561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at gz = 3.14 and gz = 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at gz = 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochromeb (b L orb 566) of the mitochondrial complex III, indicating that the gz = 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid- and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.

Electron spin resonance of microsomal cytochromes: Correlation of the amount of CO-binding species with so-called microsomal Fex in microsomes of normal tissues and liver microsomes of Sudan III-treated animals

Abstract A correlation has been established on intact microsomes of various tissues between the amount of CO-binding pigment (P-450) and microsomal Fex, which was designated as a component of rabbit liver microsomes with an ESR signal of low-spin type hemoprotein. This correlation also holds with vertebrate microsomes in which the enzyme contents were raised by injection of Sudan III into the animals. The correlation seen with intact microsomes is no longer observed after treatment with deoxycholate or alkaline, which readily destroys P-450. In the microsomes of bovine adrenal medulla a hemoprotein exists with an α-band at 559 mμ but without an ESR signal or CO-binding character.

Relationship between the interconversion of cytochrome P-450 and P-420 and its activities in hydroxylations and demethylations by P-450 oxidase systems

Abstract The relationship between the interconversion of microsomal cytochrome P-450 and P-420 and the hydroxylation and demethylation activities of the P-450 oxidase system was studied. The hydroxylation and demethylation activities could only be partly restored by complete reconversion of P-420 to P-450 by addition of polyols by dilution or by washing. The P-420 produced by detergents, monohydric alcohols or anilines did not retain hydroxylation and demethylation activities. Cytochrome P-450 is not rate limiting in the overall reaction for hydroxylation and demethylation of compounds such as anilines, nitroanisoles and aminopyrine. The substrate specificities of the hydroxylations and demethylations by the P-450 oxidase systems of different tissues differ. The rate of hydroxylation of anilines at the p-position was influenced by not only the hydrophobic character, but also by electronic and steric effects of the substrate. The magnitude of the spectral changes of the microsomes induced by the substrate corresponded to the ESR signal height at gm = 2.25 of microsomal Fex rather than to the absorption peak of the CO complex of P-450 at 450 mμ. The hydroxylation and demethylation activities were observed with either NADPH or NADH. Evidence is presented that microsomal NADPH-cytochrome c reductase is reduced by NADH and the Km for the reductase with NADH is greater than the Km for NADPH.

The electron spin resonance and absorption spectra of microsomal cytochrome P-450 and its isocyanide complexes

Abstract The absorption spectra of oxidized P-450-isocyanide complexes were the same in difference spectra irrespective of the isocyanide derivative tested. However, with these reduced P-450-isocyanide complexes, absorption at 455 mμ increased, and that at 430 mμ decreased, with increasing carbon atom number of the isocyanide derivative at a definite pH. The same changes were seen with individual complexes with increasing pH. The dissociation constants of oxidized P-450-isocyanide complexes decreased with increase in carbon atom number of the isocyanide. These results were confirmed by electron spin resonance (ESR) spectroscopy. However, the dissociation constants of reduced P-450-isocyanide complexes were essentially identical and the dissociation constants of the oxidized and reduced P-450-isocyanide complexes were little affected by pH. The oxidized P-450-isocyanide complexes gave magnetically specific ESR signals. The orbital energy differences of d e orbitals of the heme iron of the complexes increased with increase in the carbon atom number of the isocyanide. Purified P-450 and its isocyanide complexes were rapidly reduced by a ferredoxin-NADP + reductase system.

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.

The role of the hydrophobic bonding in P-450 and the effect of organic compounds on the conversion of P-450 to P-420

Abstract The conversion of cytochrome P-450 to P-420 by organic solvents was studied. The transition concentrations of ureas used for conversion of P-450 to P-420 were, in decreasing order: urea, N-methylurea, 1,1-dimethylurea, ethylurea, tetramethylurea, 1,3-diethylurea. Similar results were obtained with amides. The transition concentrations of anilines and phenols closely paralleled the π values of the organic compounds. To maintain the structure which gives P-450 its unusual properties in comparison with other known hemoproteins, the hydrophobic bonding in P-450 appears to play an important role in that it binds the heme plane to the apoprotein.

Preparation and physicochemical properties of functional hemoprotein P450 from mammalian tissue microsomes

Abstract Functional hemoprotein P450 particles were isolated from rabbit liver and pig and bovine adrenocortical microsomes by tryptic digestion in 0.1 M potassium-phosphate buffer (pH 7.0) containing 2 mM EDTA and 20% (v/v) glycerol. The optical and magnetic properties of the particles were studied at three widely different temperatures. The absolute absorption spectra of functional hemoprotein P450 of liver microsomes in the oxidized and reduced forms in the visible region had absorption peaks at 360, 414, 532 and 568 mμ in the oxidized form, at 416 and 555 mμ in the reduced form and at 450 and 555 mμ in the CO complex form at room temperature. The absolute absorption spectra of hemoprotein P450 in the visible region at the temperature of liquid N 2 had absorption peaks at 350, 414, 538 and 570 mμ in the oxidized form, at 415 and 550 mμ in the reduced form and at 447 and 550 mμ in the CO complex form. The electron paramagnetic resonance (EPR) spectrum of oxidized hemoprotein P450 at the temperatures of liquid N 2 and liquid H 2 showed a low spin form, and its g galues were identical with those of hemoprotein P450 of whole microsomes, whereas the EPR spectrum of hemoprotein P420 which had been highly purified by treatment with snake venom showed a pure high spin form at these temperatures. The same results were obtained with preparations of hemoprotein P450 from pig and bovine adrenocortical microsomes. The light absorption difference spectra in the visible region of the hemoprotein P450 complex with nitrosobenzene showed peaks at 452–453 and 554–558 mμ in the oxidized form and at 422, 455, 520 and 577 mμ in the reduced form. The absorption spectra of both forms changed with the pH. the absorption spectra of complexes of hemoprotein P450 with imidazole or triazole derivatives were also investigated. The absorption spectra of the reduced hemoprotein P450-triazole complex, unlike that of the complex of reduced hemoprotein P450 with imidazole, showed splitting of the Soret bands at 425 and 445 mμ, and these were pH dependent. On the other hand the Soret band of the oxidized hemoprotein P450-triazole complex showed neither splitting nor pH dependence. The unique structure of hemoprotein P450 is not formed only on reduction, for even the oxidized form has a unique structure. The EPR spectra of oxidized hemoprotein P450 complexes with nitrogenous ligands were studied. The optical and magnetic properties of hemoprotein P450 and its complexes with nitrogenous ligands were compared withi those of hemoprotein P420 and other hemoproteins.

Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase of pig adrenocortical mitochondria.

NADPH-adrenodoxin reductase is a component of an adrenodoxin-linked cytochrome P-450 mixedfunction oxidase system of adrenocortical mitochondria, and many steroids are hydroxylated by this mixed-function oxidase system [l] . We have reported previously on crystalline NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria [2,3] . This communication describes the crystallization of pig NADPH-adrenodoxin reductase from pig adrenocortical mitochondria by two methods and the properties of the NADPH-adrenodoxin reductase.

Cytochrome b5 and co-binding cytochromes in the Golgi membranes of mammalian livers

Abstract Cytochrome b5 and cytochrome P-450 are present in the Golgi membranes of mammalian livers. In normal rabbit liver, the Golgi membranes have a similar cytochrome b content on a protein basis to the smooth-surfaced and rough-surfaced microsomes,but their cytochrome P-450 content is very low. The cytochrome P-450 of the Golgi membranes was induced considerably by injections of sodium phenobarbital. On the other hand,on treatment of rabbits with 3-methylcholanthrene, cytochrome P-448 was induced not only in the smooth-surfaced and rough-surfaced microsomes, but also in the Golgi membranes of the liver.