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H H Ruf

Publications and source records attributed to H H Ruf.

At least 37 records · Page 2Linked to original sources
PubMed1980

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions.

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.

Alcohol Oxidoreductases↗
PubMed1979

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.

Animals↗
PubMed1979

Models for ferric cytochrome P450. Characterization of hemin mercaptide complexes by electronic and ESR spectra.

Hemin coordinated with mercaptide sulfur as fifth ligand and various sixth ligands were investigated as models for cytochrome P450 in its native ferric low-spin state and its ligand complexes. Mixing the hemin with its ligands below -60 degrees C prevented the reduction of the hemin by mercaptide and made it possible to characterize each sample both by electronic and ESR spectra. Excess of mercaptide formed hemin-dimercaptide complexes with hyperporphyrin spectra with two Soret bands around 380 and 370 nm. The second mercaptide could be exchanged by other ligands with hydroxyl, phosphine, thioether, isocyanide, amine, imidazole, and pyridine groups. The comparison of these spectral data with cytochrome P450 substantiates mercaptide as the fifth ligand and makes a hydroxyl group a more likely candidate for the native sixth ligand than an imidazole group.

Binding Sites↗
PubMed1979

Model systems for the coordination chemistry of cytochrome P-450.

Studies on model complexes have supported the presence of a mercaptide as the fifth ligand of cytochrome P-450 monooxygenases. When alcohol or thiol ligands are added to the sixth coordination position of a five-coordinated 4-nitrobenzene thiolate complex of FeIII protoporphyrin IX dimethyl ester chloride low spin complexes with optical and EPR-spectra very similar to cytochrome P-450 are obtained. From a comparison with all ligands of cytochrome P-450 and the model complexes it is concluded that a hard ligands must occupy the sixth coordination position of cytochrome P-450. An imidazole group is less likely, also in view of the ligand field parameters. The significance of the fifth and sixth ligand of cytochrome P-450 is discussed with respect to the monooxygenase mechanism.

Cytochrome P-450 Enzyme System↗
PubMed1977

Spectral evidence for a liver mitochondrial cytochrome P450 involved in bile acid metabolism.

Optical difference spectroscopy of liver mitochondria has revealed the presence of a cytochrome P450 species by its ligand reactions with carbon monoxide, metyrapone and diethylphenylphosphine. Its concentration of 0.15 nmol/mg mitochondrial protein is high enough to be detectable by ESR also. A microsomal contamination of the mitochondria could be excluded. Mitochondrial cytochrome P450 forms an enzyme-substrate complex with 5beta-cholestane-3alpha, 7alpha, 12alpha-triol with Ks value very similar to the Km value of the 26-hydroxylation of this substrate. This supports the existence in liver mitochondria of a cytochrome P450-dependent 26-monooxygenase for bile acid precursors, as previously postulated by us on the basis of a photochemical action spectrum.

Animals↗
PubMed1976

Binding of nitroxide stearate spin labels to bovine serum albumin.

1. 12-Nitroxide stearate binds to bovine serum albumin at about four independent and equivalent binding sites with an association constant of about 10(6) M-1. The binding at these high affinity binding sites is significantly reduced by addition of unlabeled stearate. These data suggest that nitroxide stearates probe the high affinity binding sites for long-chain fatty acids. 2. Qualitative analyses of the ESR spectra of 5-, 12- and 16-nitroxide stearate bound to bovine serum albumin and measurements of the interaction of these compounds so bound with ferricyanide ion provide a rough description of the binding site as follows: the polar headgroup of the spin-labeled fatty acid is rigidly fixed, but fairly accessible to paramagnetic ions. The middle part of the hydrocarbon chain of bound stearate spin label also is rigidly fixed but differs in being shielded from the solvent, presumably by a hydrophobic cleft. The methyl terminus shows greater motion, appearing to move within a narrow cone, and also appears to be somewhat accessible to paramagnetic ions.

Binding Sites↗
PubMed1976

Binding of a metyrapone spin label to microsomal cytochrome P-450.

Probing of the active site of microsomal cytochrome P-450 was carried out with a spin label derived from 2-methyl-1,2-bis(3-pyridyl)-1-propanone (metyrapone). Its optical binding spectra to cytochrome P-450 resemble the spectra with metyrapone. The electron paramagnetic resonance spectrum of the spin label in microsomes indicates binding with strong immobilization. Since the apparent optical and EPR binding constants agree very well (Ks approximately 2-10(-5) M), and metyrapone is found to displace the spin label, we conclude, that the spin label binds to the active site of cytochrome P-450. Addition of detergents or an increase in temperature mobilizes the bound spin label slightly. The EPR signal accounts for only 60% of the bound spin label due to its dipolar interaction with the low-spin ferric heme of cytochrome P-450. From this finding, the distance between nitroxide and iron is evaluated to be 11 A. This supports the model that one of the pyridine nitrogens of metyrapone is coordinated to the iron of cytochrome P-450. The bound spin label shows virtually no interaction with ferricyanide as if the active site of cytochrome P-450 is a hydrophobic pocket not accessible to ions.

Animals↗
PubMed1976

Binding of thiols to microsomal cytochrome P-450.

Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.

Animals↗
PubMed1975

Ligand binding of organic sulfides to microsomal cytochrome P-450.

Octyl methyl-, butyl methyl- and pentamethylene sulfide react with about 50% of oxidized cytochrome P-450 in liver microsomes from phenobarbital-pretreated rats by formation of optical difference spectra with maxima at 435 and 552 nm and concomitant shifts in the electron paramagnetic resonance spectrum. Reduction by NADPH or sodium dithionite yielded a Soret absorption band at 449 nm and alpha and beta bands at 573 and 545 nm, respectively. The ligand metyrapone and the substrate n-octane competitively inhibited the formation of these difference spectra and pentamethylene sulfide was a strong competitive inhibitor of the 0-deakylation of 7-ethoxycoumarin. These results indicate a direct ligand binding of the sulfides to cytochrome P-450 with concomitant blocking of the hydrophobic substrate binding site. Some sulfides did not interact as ligands but as substrates, in variation, however, with the source of microsomes.

Animals↗