Ferric Cytochrome P-450 Research Articles

Letter: A model for the carbonyl adduct of ferrous cytochrome P450.

Letter: A model for the carbonyl adduct of ferrous cytochrome P450.

A Model Compound Study of the CO-Adduct of Cytochrome P-450

Abstract A model compound was prepared having the same optical properties in the Soret and visible regions as the CO-adduct of ferrous cytochrome P-450. Obligate for the formation of this spectral species absorbing at 450 nm (emm = 93) and 555 nm (emm = 23) in a dimethylsulfoxide-ethanol solvent system was the presence of ferrous heme, CO, thiol, and a strong base. Decreasing the dielectric constant of the solvent, which would be equivalent to increasing the hydrophobicity of the heme environment in the protein, causes a small shift in the Soret absorption to the blue with no change in emm. The absence of thiol or strong base resulted in the formation of typical heme·CO absorptions at 413 nm (emm = 150), 535 nm (emm = 25), and 566 nm (emm = 30). It is concluded that the sulfur-containing axial ligand in the model compound and also in ferrous cytochromes P-450·CO is a mercaptide anion.

Superoxide anion production by the autoxidation of cytochrome P450 cam

Chemiluminescence occurs on autoxidation of oxygenated ferrous cytochrome P450 cam and is abolished by reagents that scavenge free radicals, by superoxide dismutase and singlet oxygen quenchers. We attribute the chemiluminescence to the decay of an excited singlet oxygen which arises from a superoxide anion radical precursor.

Pseudomonas putida cytochrome P-450. Binding of a spin-labeled analog of the inhibitor metyrapone

A spin-labeled analog of metyrapone, 1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl nicotinate, is similar to metyrapone in its effect on the absorbance spectrum of camphor-free ferric cytochrome P-450, shifting the Soret band from 418 to 420 nm. Spectrophotometric studies demonstrate that binding of the spin-labeled molecule is competitive with substrate (camphor) binding and yield a value of 4.1×105 m −1 for the equilibrium association constant Ka of the spin label-enzyme complex. Addition of cytochrome P-450 to an aqueous solution of the spin label considerably diminishes the EPR signal of the free, or unbound, spin label, which can be restored to a large extent by subsequent addition of camphor or metyrapone. The value of Ka determined from EPR titrations is consistent with that cited above. The EPR signal of the spin label bound to cytochrome P-450 is that of an immobilized nitroxide species in proximity to the paramagnetic ferric cation of the hemeprotein. From EPR data, the distance between these two paramagnetic species bound to the same enzyme molecule has been determined. The experimental results indicate that the spin label is directly coordinated through its pyridine nitrogen atom to the heme iron of cytochrome P-450 and that metyrapone binds similarly.

Formation and ligand binding of the fluorenyl carbanion by hepatic cytochrome P450

Fluorene interacts with cytochrome P450 from phenobarbital-pretreated rats by formation of an enzyme-substrate complex. Reduction of the fluorene-cytochrome P450 complex, after short exposure to NADPH and oxygen, causes the formation of a new spectral species with absorption bands at 446, 540, and 568 nm. The following results indicate that this spectral change is due to ligand binding of the fluorenyl carbanion to reduced cytochrome P450: 1. 1. The carbanion can be identified by its characteristic absorption at 374 nm. 2. 2. 3H is incorporated into the 9-position of fluorene after incubation with microsomes, NADPH, and oxygen. 3. 3. In the presence of 14CO 2, 14C-labeled fluorenyl-9-carboxylic acid is formed. 4. 4. The formation of a carbanion and its ligand binding to reduced cytochrome P450 explains the spectral similarity to the metyrapone binding spectrum. Since metyrapone also inhibits the carbanion formation a binding to a common ligand position is very likely. This is further supported by the close quantitative correlation of the 446 nm peak heights with fluorene and metyrapone in different microsomal prepations. It is concluded that the active oxygen can abstract a proton from CH-acidic compounds. The carbanion formed is stabilized by the ferrous cytochrome P450 at the hydrophobic active site of the enzyme.

Linear electric field-induced shifts in electron paramagnetic resonance: a new method for study of the ligands of cytochrome P-450.

The linear electric field shift in paramagnetic resonance has, for the first time, been observed in frozen solutions. The magnitude of the shift parameters, measured at g(max) of the electron paramagnetic resonance absorption envelope, has been used to characterize lowspin ferric heme mercaptide complexes where the nonmercaptide ligand was varied. The magnitude of the shift provides a measure of the difference between the crystal field contribution of the mercaptide and nonmercaptide ligands. In low-spin ferric cytochrome P-450 of rat liver, the presence of an electric field shift indicates a deviation from inversion symmetry and proves that the axial ligands to the heme are not the same. From the magnitude of the shift and its dependence upon the angle between applied electric and magnetic fields, it is suggested that the non-axial ligand to the heme has a crystal field contribution greater than imidazole and smaller than guanidine.

Carbon monoxide binding by Pseudomonas putida cytochrome P-450

The interaction of carbon monoxide with Pseudomonas putida cytochrome P-450 is affected by the substrate camphor. In the presence of camphor, the equilibrium constant for the reaction with carbon monoxide is 2.6 × 10 5 m −1 while in the absence of camphor the equilibrium constant is 2.2 × 10 6 m −1. The second-order reaction rate constant for the reaction of carbon monoxide with the camphor complex of ferrous cytochrome P-450 is 3.8 × 10 4 m −1 sec −1, while in the absence of camphor the second-order reaction rate constant is 5.1 × 10 6 m −1 sec −1. The results presented support the conclusion that camphor is bound to ferrous cytochrome P-450 at a site which markedly modifies the carbon monoxide (oxygen) binding site on the heme iron atom.

Electron paramagnetic resonance and temperature dependent spin state studies of ferric cytochrome P-450 from Rhizobium japonicum

Cytochrome P-450 as isolated from Rhizobium japonicum can be fractionated into components which are almost completely high spin and almost completely low spin, respectively. The EPR and optical spectra of low spin forms are similar to those for low spin ferric cytochromes P-450 from other sources suggesting that in the low spin Rhizobium proteins at least one of the heme ligands is the same. The addition of phenobarbital to low spin Rhizobium cytochrome P-450 changes the optical spectrum ( λ max = 535,566nm) studied at room semperature to that of a high spin form ( λ max = 505,645 nm). Lowering the temperature to near 80 °K diminishes the spectral features of this high spin form while shifting the spectrum back to that of low spin protein. The low temperature (~1.6 °K) EPR spectrum of phenobarbitaltreated low spin cytochrome P-450 shows no new high spin ferric protein, while EPR absorptions ascribable to a low spin ferric phenobarbital bound form are observed. It is concluded that the spin state of phenobarbital-treated cytochrome P-450 is temperature dependent and this accounts for the absence of an EPR spectrum of high spin cytochrome P-450 in mammalian liver microsomes from phenobarbital-treated animals.

Pseudomonas putida cytochrome P-450: Characterization of an oxygenated form of the hemoprotein

When ferrous cytochrome P-450, purified from Pseudomonas putida, was mixed with oxygen in the presence of its organic substrate, d-camphor, a new spectrally distinct species was observed whose absorption spectrum had maxima at 355, 418 and 555 nm. The absorption spectrum of this new species was essentially unchanged by cooling the reaction mixture to the temperature of liquid nitrogen. The new spectral species is rather stable in the absence of the iron-sulfur protein, putidaredoxin, a physiological electron donor for cytochrome P-450 and can be reversibly transformed into carbon monoxy-cytochrome P-450 by varying the relative partial pressures of oxygen and carbon monoxide. No electron paramagnetic resonance signal attributable to the new species could be detected between 500 and 4000 gauss at either 10.5 or 77 ° K. Oxygen, camphor and the ferrous cytochrome are required for the formation of the new spectral species. The reaction is first order with respect to both oxygen and cytochrome P-450 concentrations and the second order reaction rate constant is 7.7 × 10 5 m −1 sec −1 at 4 °. The formation of the new species proceeded without detectable intermediates. The dissociation of oxygen from the oxygenated form is first order with a rate constant of 1.1 sec −1 at 4 °. The new spectral species undergoes slow decomposition to the ferric form of the hemoprotein and is most stable in the pH range 7–8 and at temperatures below 10 °. The decomposition of the ferrous oxygen complex to the ferric form is first order with respect to the oxygenated species (rate constant = 10 −4 sec −1 at pH 7.4 and 4 °) and also proceeds without detectable intermediates. The rate of transformation to the ferric form is increased by increasing the hydrogen ion concentration, raising the temperature, or increasing the ionic strength of the reaction medium. Based on these findings, the new spectral species is interpreted to be an oxygenated form of cytochrome P-450, presumably a ternary complex of oxygen, camphor, and ferrous cytochrome P-450.

Ethyl isocyanide complexes of bacterial cytochrome P-450

The spectral properties of the ethyl isocyanide complexes of the soluble, highly purified cytochrome P-450, isolated from Pseudomonas putida, are reported. In the presence of both camphor and potassium ions, the ethyl isocyanide complex of reduced bacterial cytochrome P-450 exhibits a single Soret absorption maximum at 453 nm, and the magnitude of this maximum is unaffected by changes in pH between 6.0 and 8.0. The Soret absorption maximum of the ethyl isocyanide complex of reduced bacterial cytochrome P-420 is at 432 nm; an absorption maximum in the 430 nm region is observed with bacterial cytochrome P-450 only under conditions which promote its conversion to a type of cytochrome P-420. As a ligand of the heme iron of bacterial cytochrome P-450, ethyl isocyanide binds more tightly to the ferrous state than the ferric state, and the “apparent” K s of ferrous cytochrome P-450 for ethyl isocyanide is 9.0 × 10 −6 m in the absence of camphor. The finding that camphor competes very effectively with the binding of ethyl isocyanide to reduced cytochrome P-450 suggests that the camphor binding site is near the heme iron.

Studies of the oxidation state of partially purified adrenal cortex mitochondrial cytochrome P-450 and difference spectra induced by deoxycorticosterone and metopirone

Cytochrome P-450 prepared from extracts of adrenal gland cortex mitochondrial acetone powder exists in the oxidized state as it will not complex with carbon monoxide. Both deoxycorticosterone and Metopirone will induce spectra in the preparation in the absence of oxygen and reductase components. Metopirone displaces the deoxycorticosterone-induced difference spectrum of the cytochrome P-450 preparation, but excess deoxycorticosterone does not influence the Metopirone-induced spectrum suggesting different complexing mechanisms for the two compounds. The observations are consistent with the concept that the steroid-induced spectrum is the result of a direct complexing with ferric cytochrome P-450, or a closely associated companion pigment dependent upon the ferric state of cytochrome P-450. Current observations indicate experimentally separable reaction steps in the overall system of 11β-hydroxylation.