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At least 19 recordsLinked to original sources
PubMed1975

An interpretation of the three line EPR spectrum of nitric oxide hemeproteins and related model systems: the effect of the heme environment.

The EPR spectra of the nitric oxide (NO) derivatives of structurally perturbed Fe (II) hemeproteins show various patterns, all of which are characterized by the conspicuous three-line hyperfine splitting due to 14NO, in contrast to that of the native proteins. For the purpose of obtaining structural information from these three line spectra, the model systems were studied, which consist of NO, heme (or TPP-Fe(II), where TTP means alpha, beta, gamma, delta-tetraphenylporphine) and the nitrogenous base, pyridine or quinoline, which, respectively, give the native type or the three line (perturbed type) EPR spectrum. The ring proton paramagnetic shift of quinoline in this system shows that it is not coordinated to NO-TPP-Fe(II) as a normal axial ligand, in contrast to pyridine which gives the shift pattern of the ordinary axial ligation. This observation suggests that in the NO-hemeproteins some perturbations of the protein structure cause the rupture or distortion of the bond between the imidazole nitrogen on the fifth coordination site and the heme iron, resulting in the three line spectrum. The EPR study of the model systems, the pentacoordinated complex, NO-heme and NO-TPP-Fe(II), in various media revealed that the pentacoordinated species indeed exhibits, depending upon its environment, a variety of spectra, which closely reproduce the three line patterns observed in the perturbed proteins and some related model systems. Such spectral variation can be attributed to the difference in the degree of quenching the internal motion and/or the structural heterogeneity caused by molecular environment.

Electron Spin Resonance Spectroscopy↗
PubMed1975

Pseudomonas putida cytochrome P-450. The effect of complexes of the ferric hemeprotein on the relaxation of solvent water protons.

With pulsed nuclear magnetic resonance techniques, the effects of various complexes of ferric cytochrome P-450 on the relaxation rate of bulk solution water protons have been determined. For the camphor, metyrapone, and 4-phenylimidazole complexes, the experimental results are consistent with outer sphere relaxation effects. However, for the substrate-free enzyme, the magnitude and temperature dependence of the paramagnetic relaxation effects indicate the presence of exchangeable protons in the coordination sphere of the heme iron atom. The exchange rate (9.3 x 10(4) S-1 at 25 degrees) and the thermodynamic activation parameters for the exchange process are very similar to those of acid metmyoglobin and acid methemoglobin, suggesting that a water molecule, and not an amino acid residue of the protein, coordinates to the ferric cation of the enzyme in the absence of added substrate or ligands. From the equations appropriate for coordination sphere protons, the distance between these protons and the ferric heme cation was evaluated as 2.1 A, which further supports the interpretation. These experimental results demonstrate that the solvent accessibility of the ferric cation of substrate-free cytochrome P-450 is significantly reduced by the binding of substrate or nitrogenous ligands to the hemeprotein.

Binding Sites↗
PubMedDate not supplied

Assimilatory nitrate reductase of Rhodopseudomonas capsulata AD2: a molybdo-hemeprotein.

The assimilatory nitrate reductase of the phototrophic bacterium Rhodopseudomonas capsulata strain AD2 was purified to homogeneity by a combination of ammonium sulfate fractionation, chromatography on DEAE-cellulose and isoelectric focusing (isoelectric point of 4.8). The purified enzyme was active only with reduced viologen dyes or reduced flavin as electron donors. Contrary to other bacterial assimilatory nitrate reductases, the enzyme was not inhibited by chlorate, but rather accepted this substance as an alternate substrate. The molecular weight of the enzyme was 185,000 dalton as determined by gelfiltration. Subunit analysis by sodium dodecyl sulfate (SDS) gel electrophoresis yielded a single protein band with a molecular weight of 85,000 dalton,, suggesting that the enzyme was composed of two identical subunits. The nitrate reductase contained 0.8 g-atoms molybdenum per 1.85 x 10(5) g protein and exhibited absorption maxima at 418, 523 and 552 nm in the reduced state (dithionite as reductant). The nitrate reductase of Rps. capsulata AD2 is the first prokaryotic enzyme of the assimilatory type that has been shown to contain heme.

Hemeproteins↗
PubMed1978

Restoration of the responsiveness of purified guanylate cyclase to nitrosoguanidine, nitric oxide, and related activators by heme and hemeproteins. Evidence for involvement of the paramagnetic nitrosyl-heme complex in enzyme activation.

Purification of soluble guanylate cyclase activity from rat liver resulted in loss of enzyme responsiveness to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitroprusside, nitrite, and NO. Responses were restored by addition of heat-treated hepatic supernatant fraction, implying a requirement for heat-stable soluble factor(s) in the optimal expression of the actions of the activators. Addition of free hematin, hemoglobin, methemoglobin, active or heat-inactivated catalase partially restores responsiveness of purified guanylate cyclase to MNNG, NO, nitrite, and nitroprusside. These responses were markedly potentiated by the presence of an appropriate concentration of reducing agent (dithiothreitol, ascorbate, cysteine, or glutathione), which maintains heme iron in the ferro form and favors formation of paramagnetic nitrosyl . heme complexes from the activators. High concentrations of heme or reducing agents were inhibitory, and heme was not required for the expression of the stimulatory effects of Mn2+ or Mg2+ on purified guanylate cyclase. Preformed nitrosyl hemoglobin (10 micron) increased activity of the purified enzyme 10- to 20-fold over basal with Mn2+ as the metal cofactor and 90- to 100-fold with Mg2+. Purified guanylate cyclase was more sensitive to preformed NO-hemoglobin (minimally effective concentration, 0.1 micron) than to MNNG (1 micron), nitroprusside (50 micron), or nitrite (1 mM). A reducing agent was not required for optimal stimulation of guanylate cyclase by NO-hemoglobin. Maximal NO-hemoglobin-responsive guanylate cyclase was not further increased by subsequent addition of NO, MNNG, nitrite, or nitroprusside. Activation by each agent resulted in analogous alterations in the Mn2+ and Mg2+ requirements of enzyme activity, and responses were inhibited by the thiol-blocking agents N-ethylmaleimide, arsenite, or iodoacetamide. The results suggest that NO-hemoglobin, MNNG, NO, nitrite, and nitroprusside activate guanylate cyclase through similar mechanisms. The stimulatory effects of preformed NO-hemoglobin combined with the clear requirements for heme plus a reducing agent in the optimal expression of the actions of MNNG, NO, and related agents are consistent with a role for the paramagnetic nitrosyl . heme complex in the activation of guanylate cyclase.

Animals↗
PubMed1975

Partial purification and separation of multiple forms of cytochrome. P-450 and cytochrome P-448 from rat liver microsomes.

1. Partial purification of liver microsomal cytochrome p-450 results in the separation of two forms of cytochrome p-450 from phenobarbital-treated rats and two forms of cytochrome p-44, from 3-methylcholanthrene-treated rats. 2. Each of the four cytochrome fractions had different spectral properties (absolute spectra, CO differences spectra, and ethylisocyanide difference spectra). 3. The hemeprotein in fractions which elute from a DEAE-cellulose column at 100 mKM KCl fraction IV B) are more highly purified than the hemeproteins (fraction IV A) that elute in the column volume. 4. The more highly purified cytochrome fractions (IV B) contain 9-11 moles of cytochrome P-450 or P-448 per mg protein (an approximately 5-7 fold purification over microsomes) and are enzymatically active in the metabolism of a variety of substrates when combined with lipid and NADPH-cytochrome c reductase. These hemeprotein fractions are free of cytochrome b5 and NADPH-cytochrome c reductase, and the hemeproteins are purified approximately 100-fold with respect to phospholipid. The cytochrome P-450 and P-448 are virtually free of epoxide hydrase.

Animals↗
PubMed1976

Multiple forms of rat liver cytochrome P-450. Immunochemical evidence with antibody against cytochrome P-448.

Purified hepatic cytochrome P-448 from 3-methylcholanthrene-treated rats was used to produce antibody in rabbits. The cytochrome P-448 antibody (IgG fraction) isolated from immune rabbit serum is quite specific and precipitates purified rat liver cytochrome P-448 at low antibody to protein ratios when assayed by the Ouchterlony double diffusion technique. Purified hepatic cytochrome P-450 from phenobarbital-treated rats cross-reacts poorly with the cytochrome P-448 antibody as do purified rabbit hepatic cytochrome P-448 and P-450. No cross-reaction is observed with purified cytochrome P-450 from beef adrenal mitochondria or from Pseudomonas putida in Ouchterlony double diffusion experiments. The cytochrome P-448 antibody produces a single distinct precipitin band with purified rat cytochrome P-448. In contrast, purified liver cytochrome P-450 from phenobarbital-treated rats gives three precipitin bands, all of which contain hemeprotein as judged by benzidine staining. At least two of the three precipitin bands are immunochemically different from the precipitin band formed with cytochrome P-448. When added to the reconstituted system, the cytochrome P-448 antibody inhibits purified rat cytochrome P-448- and P-450-supported N-demethylation of benzphetamine, O-deethylation of ethoxycoumarin, hydroxylation of benzo[a]pyrene, and the hydroxylation of testosterone at the 6beta, 7alpha, and 16alpha positions. Antibody inhibits cytochrome P-448-supported metabolism more than cytochrome P-450-supported metabolism except for benzo[a]pyrene hydroxylation at low antibody to hemeprotein ratios. In addition, the pattern and extent of inhibition of the cytochrome P-450 system depends on the substrate used, suggesting that multiple forms of the hemeprotein are present in the purified preparation from phenobarbital-treated rats. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicate that (a) cytochrome P-448 from 3-methylcholanthrene-treated rats is immunochemically different from cytochrome P-450 from phenobarbital-treated rats, and (b) there appear to be at least three hemeprotein forms in the purified cytochrome P-450 preparation from phenobarbital-treated rats.

Animals↗
PubMed1975

Multiple forms of cytochrome P-450 in phenobarbital- and 3-methylcholanthrene-treated rats. Separation and spectral properties.

Multiple forms of liver microsomal cytochrome P-450 isolated from immature male rats pretreated with phenobarbital or 3-methylcholanthrene are described. Afraction of low specific content (Fraction A. 1.7 TO 4.0 nmol of cytochrome P-450 per mg of protein) and a fraction substantially purified (Fraction B, 9.0 TO 11.0 NMOL of cytochrome P-450 per mg of protein) are obtained by DEAE-cellulose chromatography of a partially purified cytochrome P-450 preparation in the presence of Emulgen 911. Shifts in the absorption maxima in the CO-reduced and ethyl isocyanide difference spectra are observed in the fractions derived from 3-methylcholanthrene-treated rats. The fractions derived from phenobarbital-treated rats exhibit different 455:430 ratios and pH intercepts in the ethyl isocyanide difference spectra. The absolute oxidized spectra and n-octylamine binding spectra at room temperature and EPR analysis at the temperature of liquid helium characterize all the fractions, except the Fraction A from 3-methylcholanthrene-treated rats, as low spin ferric hemeproteins. The A hemeprotein fractions from both 3-methylcholanthrene- and phenobarbital-treated rats have poor catalytic activity for the metabolism of benzphentamine and 3,4-benzo-[a]pyrene in comparison to the B hemeprotein fractions which may be due to the presence of a high concentration of Emulgen 911 in the A fractions. However, the presence of Emulgen 911 cannot account for the spectral differences among the fractions.

Amines↗
PubMed1975

Spectroscopic properties of ferrous heme complexes of sterically hindered ligands.

Mesoheme IX complexes of sterically hindered ligands 2-methylimidazole, tert-butylamine and 2-methylpyridine in aqueous glycerol solutions are characterized by broad visible absorption spectra at ambient temperature exhibiting close similarities to high-spin ferrous hemeproteins. Spectrophotometric titrations of mesoheme IX with these ligands indicate well-defined equilibria for 2-methylimidazole and tert-butylamine corresponding to the formation of penta-coordinate strong-field ligand complexes. Variable temperature spectra of these complexes from ambient to 77 degrees K exhibit a change to hemochrome spectra characteristic of the low-spin unhindered ligand complexes. Corresponding changes in the visible spectra are not observed for the high-spin hemeproteins deoxymyoglobin, horse-radish peroxidase and cytochrome ć. The appropriate utilization of these hindered ligand heme complexes as model systems for high-spin ferrous hemeproteins has been discussed.

Binding Sites↗