Amino acid sequences of cytochrome b5 from human, porcine, and bovine erythrocytes and comparison with liver microsomal cytochrome b5.

The amino acid sequences of human, porcine, and bovine erythrocyte cytochromes b5 which are soluble and present in the cytosol have been determined. In addition, the partial sequences of microsome-bound liver cytochrome b5, namely the sequence of the N-terminal region and joint region between the heme-containing and membranous part, have been established for human and porcine sources. All the cytochromes b5 from erythrocyte and liver contained N-acetylated N-termini. Of the 97 amino acid residues of erythrocyte cytochrome b5, residues 1-96 were identical with those of the liver protein of the same species. However, residue 97 (C-terminal residue) was proline for human erythrocyte cytochrome b5 and serine for the porcine protein, while residues 97 (joint region) of human and porcine liver cytochromes b5 were threonine. These findings indicate that the two forms of cytochrome b5 are encoded by two different but closely related mRNAs.

Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase.

The mechanism of hepatic NADPH-cytochrome P-450 reductase has been investigated by using a stopped-flow technique. The reduction of the oxidized native enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron eqiuvalent mechanisms. The air-stable semiquinone form (FAD-FMNH.) of the native enzyme, which is characterized by an absorption shoulder at 635 nm, is also rapidly reduced to another semiquinone form (FADH-FMNH2) by NADPH with the disappearance of the shoulder at 635 nm, but the absorbance change at 585 nm is relatively constant. The FAD moiety in the FMN-depleted enzyme is rapidly reduced by NADPH, and reduced FAD is oxidized in successive one-electron steps by O2 or potassium ferricyanide. These results indicate the possibility of intra-molecular one-electron transfer between FAD and FMN. The rate of cytochrome P-450 reduction decreases in the presence of FMN-depleted enzyme but is nearly restored to the value of the original enzyme with FMN-reconstituted enzyme. These data suggest that FAD is the low-potential flavin, which serves as an electron acceptor from NADPH. On the other hand, FMN, which is the high-potential flavin, appears to participate as an electron carrier in the process of electron transfer from NADPH to cytochrome P-450 during the mixed-function catalytic cycle.

pH dependency of kinetic parameters and reaction mechanism of beef liver catalase.

The pH-dependence of the kinetic parameters in H2O2 decomposition by beef liver catalase was investigated. At pH 7.0, the ternary complex (ESS) decomposition rate was about 100 times faster than ESS formation (42 microM H2O2), and the value of the Michaelis constant was 0.025 M. From ethanol competition experiments, two different proton dissociation constants of the enzyme (pKe1 = 5.0, pKes2 = 5.9) were obtained for the binding of first and second H2O2 molecules. Another pKa value (pKes1) of 4.2 was obtained from the pH dependence of overall rate constant (ko). The reaction mechanism of catalase was discussed in relation to these ionizable groups.

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.

Circular dichroism and magnetic circular dichroism studies on methemoglobin and its derivatives.

CD and MCD spectra of human methemoglobin and its derivatives were studied with special reference to the relation of CD and MCD spectra to the magnetic moments. The intensities of MCD peaks of methemoglobin derivatives at 270 nm were inversely proportional to their magnetic susceptibility, and there was a linear relationship between magnetic ellipticity and magnetic susceptibility. The MCD peak intensity of methemoglobin peroxide compound was close to that of methemoglobin cyanide complex, suggesting that the methemoglobin peroxide compound is in a low-spin state.

The chemical modification of beef liver catalase. V. Ethoxyformylation of histidine and tyrosine residues of catalase with diethylpyrocarbonate.

In order to elucidate the possible roles of histidine and tyrosine residues of catalase [EC 1.11.1.6] in maintaining the quaternary structure and catalatic activity, diethylpyrocarbonate modification experiments were carried out. A method for the estimation of N-ethoxyformyl (EF)-His at pH 5--7 and of O-ethoxyformyl (EF)-Tyr in alkaline solution by measuring A 242 nm (ximM = 3.2) and A278 nm (ximM = 1.16), respectively, was developed. The formation of EF-His and EF-Tyr was an electrophilic reaction and was dependent on pH, exhibiting pK values of 6.8 and 9.9, respectively. The maximal yield of EF-His at pH 6.0 was 49% of the total histidine content, but no inactivation nor unfolding of the enzyme was observed. The formation of 12 EF-Tyr residues per mole of catalase at pH 8.1 did not cause any inactivation, but the formation of 8 more EF-Tyr residues at pH 8.9 resulted in both inactivation and unfolding. Nearly complete inactivation and partial splitting of catalase were observed when 43-46 EF-Tyr residues per mole were produced at pH 10.0. More EF-His residues were formed by the reaction of diethyl pyrocarbonate with cyanoethylated (CE)-catalase monomer (subunit) than with CE-catalase tetramer. The CE-catalase tetramer and monomer were extensively O-ethoxyformylated, reaching 100% EF-Tyr formation. These results indicate that a half of the histidine residues may lie outside the protein core and that three-quarters of the tyrosine residues are probably in the protein core of the enzyme. The production of 2--3 EF-Tyr residues per mole of the monomer by ethoxyformylation at pH 7.0 was accompanied by a decrease in the magnitude of the Soret peak. A possible interaction of those tyrosine residues with porphyrin of the heme group is discussed.

Conformation of biological macromolecules. Circular dichroism and magnetic circular dichroism studies of metmyoglobin and its derivatives.

The circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of horse heart metmyoglobin and the following derivatives were measured in the Soret and near ultraviolet regions: metmyoglobin and its peroxide compound, and hydroxide, cyanide, azide, and fluoride derivatives. The heme-related CD bands in the Soret and near ultraviolet wavelength regions were altered by ligand substitution, though their relationships to the magnetic moment were quite different. In the Soret region, the CD peak had no definite relation to the magnetic moment, while in the near ultraviolet region the magnitude of the CD peak decreased with the magnetic moment. The MCD peak in the Soret and near Ultraviolet regions also varied with ligand substitution. The magnetic ellipticity decreased with the magnetic moment in both wavelength regions. There was a more quantitative correlation between the magnetic ellipticity and the magnetic moment in the near ultraviolet region than in the Soret region. Metmyoglobin peroxide compound exhibited slightly different behavior in the MCD spectrum from other derivatives. It is suggested that the heme iron of the metmyoglobin peroxide compound is in an oxidation state other than the ferric state and that the porphyrin structure of metmyoglobin may be modified by the reaction with hydrogen peroxide.