Studies of microsomal cytochrome P-450 with isocyanide spin label

Reichman, L.M.; Annaev, B; Rozantzev, E.G.

doi:10.1016/0005-2795(72)90157-2

Cited by 19 publications

(4 citation statements)

References 14 publications

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“…As to the changes induced by the type I substrates, they are explained as events occurring due to binding of hydrophobic substrates in close vicinity of the sixth heme ligand, namely, substitution of hydroxyl for the water molecule and dislocation of ligand to a more hydrophobic region (Archakov 1975). It was shown that binding of the type I substrates results in an increase in mobility of isocyanate spin probe as if an expansion of hydrophobic region around heme takes place (Raikhman et al 1972). Most probably, detergent molecules induce similar changes, increasing availability of the active site for substrates or facilitating their effective binding.…”

Section: Discussionmentioning

confidence: 98%

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

2006

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Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM(2)) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380-450 nm.

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Section: Discussionmentioning

confidence: 98%

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

2006

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“…During the course of our studies on spin-labeled myosin, we observed the selective destruction of the weakly immobilized spectral component on incubation with K3Fe(CN)6. This phenomenon could not be explained by previous studies which have shown that K3Fe-(CN)6 acts as an oxidizing agent, preventing or reversing the loss of signal brought about by chemical reduction of the spin-label (Kaplan et al, 1973;Ross & McConnell, 1975; Quintanilha & Packer, 1977), nor could it be accounted for in terms of paramagnetic broadening by K3Fe(CN)6, which results in the loss of spectral amplitude (Likhtenshtein et al, 1970;Grebenshchikov et al, 1971;Reichman et al, 1972;Likhtenshtein, 1976;Keith et al, 1977;Morse, 1977;Berg et al, 1979).…”

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confidence: 86%

A reaction involving protein sulfhydryl groups, a bound spin-label, and K3Fe(CN)6 as a probe of sulfhydryl proximity in myosin

Graceffa¹,

Seidel²

1980

Biochemistry

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The EPR spectrum of myosin labeled with an iodoacetamide spin-label shows components corresponding to weakly and to strongly immobilized labels. Upon treatment of this myosin wiith K,Fe(CN),, a chemical reaction involving the nitroxide group of the spin-label, Fe(CN):-, and a sulfhydryl group of the protein results in the selective destruction of the weakly immobilized spectral component without loss of ATPase activity. The involvement of a sulfhydryl group in this reaction is indicated by the fact that K3Fe(CN), destroys the electron paramagnetic resonance (EPR) signal of a spin-label only in the presence of a thiol-containing compound and that the signal is not lost if the sulfhydryl group is blocked. In the presence of MgADP, both the strongly immobilized component, attributable to labels bound to the SH-1 thiols of myosin, and the weakly immobilized component

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“…An isocyanide spin label has been used by Reichman et al [7] for studying microsomal cytochrome P-450.…”

Section: The Epr Signal Accounts For Only 60mentioning

confidence: 99%

“…Also by means of magnetic interactions distances from and accessibilities for paramagnetic ions can be estimated [5,6]. So this technique seems to be useful for studying the active site of cytochrome P-450, if a suitable label that binds with good affinity and high specificity to cytochrome P-450 can be found.An isocyanide spin label has been used by Reichman et al [7] for studying microsomal cytochrome P-450. …”

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confidence: 99%

Binding of a Metyrapone Spin Label to Microsomal Cytochrome P‐450

Ruf

Nastainczyk

1976

European Journal of Biochemistry

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Probing of the active site of microsomal cytochrome P-450 was carried out with a spin label derived from 2-methyl-I ,2-bis(3-pyridyl)-l -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 ( K s -2 . 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.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. The EPR signal accounts for only 60There is considerable interest in the structure of the active site of cytochrome P-450 catalyzing the monoxygenation of various substrates, which has been reviewed recently [I]. This interest arises from from two points: from the powerful activation of molecular oxygen accomplished by cytochrome P-450 and from the broad range of substrates which can be metabolized by (cytochrome P-450)-dependent monoxygenases. The activation of oxygen brings up questions concerning the polarity of the active site and its accessibility to protons and water, since model studies suggest that protons might have great influence on the active oxygen formation [2]. The geometrical relationship between the binding and catalytic sites plays an important role in the catalytic activity and substrate specificity of cytochrome P-450.The nitroxide spin label technique can provide information about the polarity of a microenvironrnent [3], and about the mobility or rotational freedom of a spin label [4]. Also by means of magnetic interactions distances from and accessibilities for paramagnetic ions can be estimated [5,6]. So this technique seems to be useful for studying the active site of cytochrome P-450, if a suitable label that binds with good affinity and high specificity to cytochrome P-450 can be found.An isocyanide spin label has been used by Reichman et al. [7] for studying microsomal cytochrome P-450. ~-Ahhreviution. EPR, electron paramagnetic resonance. TriviuINunrr. Metyrapone, 2-methyl-I ,2-bis(3-pyridyl)-l-propanone.But their spectra do not show any magnetic interaction between the spin label and the ferric heme. However, if the spin is bound coordinately to the heme iron with its isocyanide group, the resulting maximum iron-nitroxrde distance of 7 should lead to a strong magnetic interaction. Since our reinvestigation with this spin label reveals such a strong magnetic...

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Studies of microsomal cytochrome P-450 with isocyanide spin label

Cited by 19 publications

References 14 publications

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

A reaction involving protein sulfhydryl groups, a bound spin-label, and K3Fe(CN)6 as a probe of sulfhydryl proximity in myosin

Binding of a Metyrapone Spin Label to Microsomal Cytochrome P‐450

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