Comparison of the peroxidatic activity of cytochrome P-450 with other hemoproteins and model compounds.

The H2O2 dependent catalysis of cytochrome P-450 was compared with the catalytic mechanism of horse radish peroxidase, methemoglobin and iron protoporphyrin complexes. A relatively stable intermediate being comparable to compound I of horse radish peroxidase is formed in the case of iron porphyrin complexes, methemoglobin and probably cytochrome P-450. In the case of peroxidase compound II is the more stable intermediate. This could be the reason for the different catalytic properties of peroxidase on the one hand and iron porphyrin complexes, methemoglobin and cytochrome P-450 on the other hand.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

Interaction of aliphatid alcohols with cytochrome P-450 from rat liver microsomes]

The interaction of alyphatic alcohols and cyclohexanol with cytochrome P-450 in microsomes has been investigated. All alchohols induced the modified 11 type spectral changes by mixing with microsomes. These changes are characterized by lambdamax = 412 and lambdamin = 380-382 nm in difference spectra. The dissociation constants of the alcohol cytochrome P-450 complexes are determined. On this dissociation constants influence pH and Triton X-100 presence. The interaction of the alcohols with cytochrome P-450 in phosphate buffer pH = 6,0 in the detergents absence is characterized by one dissociation constant for MeOH, EtOH, n-BuOH and cyclohexanol and by two dissociation constants for i-PrOH, i-BuOH and tert.-BuOH. The interaction of the alcohols with cytochrome P-450 in Tris-HCL-buffer (pH 7.5) in the Triton X-100 presence is characterized for all above alcohols by the dissociations constants, which are described by Taft equation with coefficient rho =-1.55. This fact confirms the interaction of alcohols HO-groups with heme iron of cytochrome P-450. The scheme of interaction of alcohols with cytochrome P-450 is discussed.     

Effect of cytochrome b5 on the functional activity and conformational state of cytochrome P-450

Microsomal cytochromes P-450 and b5 were shown to form mixed complexes with the association constant of 0.24 microM in water solution. Such complex formation stabilizes cytochrome P-450 in the catalytically active conformational state characterized by increased conformational rigidity and temperature stability. This stabilization results in acceleration of the cumene hydroperoxide-dependent oxidation of p-nitroanisol catalyzed by cytochrome P-450. The thermodynamic parameters of O-demethylation of p-nitroanisol catalyzed by cytochrome P-450 and mixed haemoprotein complexes measured in water solution and in a membrane-bound state were found to be different.     

Circular dichroism studies of low-spin ferric cytochrome P-450CAM ligand complexes

Circular dichroism (CD) spectroscopy has been used to probe the active site of bacterial ferric cytochrome P-450CAM. The endogenous sixth ligand to the heme iron has been displaced by an extensive series of exogenous oxygen, nitrogen, sulfur and other neutral and anionic donor ligands in an attempt to examine systematically the steric and electronic factors that influence the coupling of the heme chromophore to its protein environment. General trends for each ligand class are reported and discussed. Both the wavelengths and the intensities of the CD bands vary with ligand type and structure. All but one of the complexes exhibit negative CD maxima in their delta and Soret bands. Comparison to ferric myoglobin-thiolate complexes indicates that the negative sign observed for the cytochrome P-450 spectra is not a property of the thiolate fifth ligand, but rather arises from a different interaction of the cytochrome P-450 heme with its protein environment. Complexes with neutral oxygen donors display CD spectra that most closely resemble the spectrum of the native low-spin enzyme. Hyperporphyrin (split Soret) cytochrome P-450 complexes with thiolates, phosphines and cyanide trans to cysteinate have complex CD spectra, reflecting the intrinsic non-degeneracy of the Soret pi pi transitions. The extensive work presented herein provides an empirical foundation for use in analyzing the interaction of heme chromophores with their protein surroundings, not only for the cytochrome P-450 monooxygenases but also for heme proteins in general.     

Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: the origin of double Soret spectra.

Trivalent oxygenated phosphorus ligands include alkyl and aryl phosphites, (RO)3P, phosphonites, (RO)2PR, and phosphinites, ROPR2. All such compounds tested, with the exception of triphenyl phosphite, interact with ferrous cytochrome P-450 and its denatured form, cytochrome P-420, to produce complexes having two peaks in the Soret region of their optical difference spectra. Careful evaluation of these spectra indicate that they arise for different reasons for each of the two cytochromes. Clear evidence shows that cytochrome P-450 is not denatured by these ligands. The high affinity of these ligands for heme iron is indicated by small Ks values. The experimental results are used to substantiate a theory of the origin of microsomal double Soret spectra and the nature of the environments available for microsomal cytochromes P-450 and P-420.     

Formation of complexes between microsomal cytochrome P-450-Fe(II) and nitrosoarenes obtained by oxidation of arylhydroxylamines or reduction of nitroarenes in situ.

A cytochrome P-450 complex exhibiting a Soret peak at 454 nm is formed by direct interaction of nitrosobenzene with NADPH-reduced rat liver microsomes in anaerobic conditions, by reaction of phenylhydroxylamine with aerobic microsomes or during nitrobenzene reduction by NADPH-reduced or dithionite-reduced microsomes. In the latter conditions, the complex formation is only transient as it is unstable to dithionite. Analogous reactions with myoglobin lead to the previously described myoglobin-Fe(II)-nitrosobenzene complex which has similar properties to those of the 454-nm-absorbing cytochrome P-450 complex. This analogy, together with the various conditions of its formation, strongly indicates that it is a cytochrome-P-450-Fe(II)-nitrosobenzene complex. The corresponding complex with the 4-chloro-nitrosobenzene ligand is formed in similar conditions. Cytochrome P-450-Fe(II) complexes with nitrosoarenes seem less stable than the previously described complexes with nitrosoalkanes.     

The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation.

We have utilized 11beta-hydroxylase activity and visible absorption spectrophotometry to detect possible complex formation among adrenodoxin reductase, adrenodoxin, and cytochrome P-450(11)beta. At low ionic strength, a 1:1 complex between adrenodoxin reductase and adrenodoxin occurs but does not support maximal rates of 11beta hydroxylation; at least 1 additional molecule of adrenodoxin in excess of the 1:1 complex is required for full hydroxylase activity. Spectrophotometric titration of a mixture of adrenodoxin reductase and cytochrome P-450(11)beta with adrenodoxin indicates sequential formation of 1:1 complexes between adrenodoxin reductase and adrenodoxin and then between a second adrenodoxin and cytochrome P-450(11beta; the adrenodoxin-cytochrome P-450(11)beta complex is only detectable when the concentration of adrenodoxin exceeds that of adrenodoxin reductase.     

The covalent-sorption reconstitution of steroid hydroxylases

The effect of covalent immobilization via free amino groups on the catalytic activity of individual components of the cholesterol side-chain cleavage and 11b-steroid hydroxylation systems (adrenodoxin reductase, adrenodoxin, cytochrome P-450scc and cytochrome P-450(11)b) as well as on that of co-immobilized protein complexes. The protein complex formation at different stages of the monooxygenase cycle (i.e., reduction, oxygenation) was followed by direct spectrophotometric monitoring of the functional state of the immobilized complexes. Cholesterol side-chain cleavage was carried out in minicolumns, using various combinations of immobilized and soluble proteins. Cytochromes P-450scc and P-450(11)b were found to retain their functional activities after immobilization via free SH-groups.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in liposomes demonstrated by combining protein rotation with antibody-induced cross-linking

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).     

Transformation of an arachidonic acid hydroperoxide into epoxyhydroxy and trihydroxy fatty acids by liver microsomal cytochrome P-450

In the absence of NADPH, the addition of an arachidonic acid hydroperoxide, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid, to liver microsomes, prepared from phenobarbital-treated rats, resulted in the formation of two major metabolites and several minor products, some of which have been purified by reverse-phase high-performance liquid chromatography. We propose the structures of the two major products to be 13-hydroxy-14,15-epoxyeicosa-5,8,11-trienoic acid and 11,14,15-trihydroxyeicosa-5,8,12-trienoic acid based on spectral characteristics and mass spectral analysis of derivatives of the compounds. A potential heterolytic cleavage product, 15-hydroxyeicosa-5,8,11,13-tetraenoic acid, was not a product of the reaction. Ferric cytochrome P-450 catalyzed the formation of these products as shown by the inability of boiled microsomes to support the reaction, the inhibition of epoxyhydroxy and trihydroxy fatty acid formation by imidazole derivatives which bind tightly to the ferric heme iron of cytochrome P-450, and the inability of carbon monoxide (which binds to ferrous P-450) and free iron chelators (EDTA and diethylenetriaminepentaacetic acid) to inhibit product formation. These results show that liver microsomal cytochrome P-450, in addition to its role in the NADPH-dependent metabolism of arachidonic acid, can utilize a hydroperoxide to produce an interesting series of potentially important arachidonic acid metabolites.     

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. F?rster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

Induction of specific cytochrome P-450 isozymes by methylenedioxyphenyl compounds and antagonism by 3-methylcholanthrene

Two methylenedioxyphenyl compounds, isosafrole (5-propenyl-1,3-benzodioxole) and an analog, 5-t-butyl-1,3-benzodioxole (BD), differ markedly as inducers of cytochrome P-450 isozymes in rat liver microsomes. Isosafrole is a mixed-type inducer, inducing P-450b, P-450c, and P-450d. In contrast, BD is a phenobarbital-type inducer, increasing P-450b, but producing little or no increase in P-450c or P-450d. Similarly, isosafrole increases the amount of translatable mRNA for P-450b, c and d, while BD induces only the mRNA for P-450b. Dimethylation of the methylene bridge carbon of BD to give 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBD) blocks the formation of NADPH-reduced Type III metabolite-P-450 complexes in vitro, and diminishes but does not abolish the ability of the compound to induce P-450b. Western blots of microsomes from isosafrole and BD-treated rat livers confirm that in contrast to isosafrole, BD does not induce P-450d or P-450c. However, the antibody to P-450d recognizes two new polypeptides (approximately 50K Mr) from sodium dodecyl sulfate-polyacrylamide gels of liver microsomes from BD-treated rats. These polypeptides are not observed in control, isosafrole, 3-methylcholanthrene (3-MC), or DBD-treated rats. They are intensified by coadministration of 3-MC with BD and may represent either modified isozyme-metabolite adducts or degradation products of P-450d. However, the polypeptides could not be generated in vitro by addition of BD to 3-MC-induced microsomes with NADPH under conditions which produced spectral metabolite complexes, or in a reconstituted system with P-450d. The two methylenedioxyphenyl compounds do not form stable metabolite complexes with the same P-450 isozymes. BD formed distinct spectral metabolite complexes in vitro with both P-450b and P-450c but not with P-450d in a reconstituted system. In contrast, isosafrole forms metabolite complexes with all three isozymes. Coadministration of 3-MC with BD blocked induction of P-450b by 80% and produced a similar repression of its translatable mRNA. This finding indicates that 3-MC type inducers not only induce certain cytochrome P-450 isozymes, but also repress synthesis of other isozymes.     

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.     

Kinetic and spectral parameters of the amines oxidative N-dealkylation with participation of the liver microsomal cytochrome P-450. Amines oxidation with organic hydroperoxides

Kinetics of demethylation of a number of amines involving hepatic microsomal cytochrome P-450 and organic hydroperoxides (tret-butyl- and cumylhydroperoxide) have been investigated. Decomposition rate constants for the substrate-cytochrome P-450-ROOH complexes have been determined in a generalized form. Activation parameters, deltaH* and deltaS*, are calculated for decomposition of the complexes. There is a linear relation between deltaH* and deltaS*: deltaH*=18.7 kcal + 333 degrees K deltaS*. Compensation relationship is characterized by the value of alpha=333 degrees K/Taverage=1.11. The nature of the limiting step in the cytochrome P-450-NADPH-O2-system and the cytochrome P-450-ROOH-system is discussed.     

The role of the hydrophobic fragment of cytochrome b5 in the interaction with cytochrome P-450

The interaction of highly purified liver microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 has been investigated by the difference and second derivative difference spectroscopy. The addition of cytochrome b5 to cytochrome P-450 results in transition of cytochrome P-450 heme iron from low to high spin state. The interaction is accompanied by the changes in the second derivative spectrum of cytochrome P-450, which point to the participation of tryptophanyl residues in this process. The hydrophilic fragment of cytochrome b5 is unable to form a complex with cytochrome P-450 as judged by the absence of the difference spectrum and any changes in the second derivative UV-spectrum of cytochrome P-450. The evidence obtained indicates that the hydrophobic tail of the cytochrome b5 molecule responsible for its binding to membrane is also indispensable for forming a functional cytochrome P-450-cytochrome b5 complex.     

Dose dependent suppression of hepatic cytochrome P-450 content by doxorubicin and Mitomycin-C: correlation with antipyrine biotransformation

Doxorubicin (DOX) and Mitomycin-C (MMC) are two anthraquinones which, when administered to rats, result in a decrease in the content of hepatic cytochrome P-450 and mixed function oxidase activities. DOX administration produced a dose-dependent immediate decrease in cytochrome P-450 content at all doses but a parallel dose-dependent decrease in the rate of antipyrine metabolite formation of the two higher doses. The lower dose of DOX produced an increase in metabolite formation and produced a less than 20% reduction in cytochrome P-450 content. MMC administration produced an immediate, modest (less than 10% of control levels) suppression of hepatic cytochrome P-450 content, and had no effect on antipyrine metabolite formation. These findings demonstrates that two drugs of the same class can produce similar suppressions of cytochrome P-450 content and that a threshold suppression of cytochrome P-450 content is needed to produce alterations in in vivo drug biotransformations.     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.