Regulation of cytochrome P4501A by protein kinase C: the role of heat shock protein70

Carbofuran is a pesticide, which is used throughout the world as a nematicide and an acaricide. This pesticide integrates into living organisms through aquatic ecosystem. In earlier report, we had demonstrated that cytochrome P4501A was induced in cultured catfish hepatocytes in response to carbofuran, which might be responsible for the detoxification of this pesticide. As the underlying signaling mechanism associated with induction and regulation of cytochrome P4501A has not yet been well defined, we therefore in the present study have investigated to identify the regulatory network of cytochrome P4501A in catfish liver or cultured hepatocytes by targeting several key signaling molecules such as phosphatidyl inositol (PI) or protein kinase C (PKC), which are critical molecules for many important pathways. PKC and heat shock protein70 (HSP70) have been shown to be induced in response to carbofuran in catfish hepatocytes. Results also indicate that induction of CYP1A is modulated by HSP70 and PKC in fish hepatocytes. Thus our data shed light on the regulation of EROD activity, which has been used as a bio-monitoring tool for measuring aquatic pollution.     

Induction of CYP1A by carbofuran in primary culture of fish hepatocytes

Carbofuran is a nematicide used in agricultural fields throughout the world. Indiscriminate use of this pesticide poses severe detrimental effects on our ecosystem. We have shown that it induces the CYP1A (cytochrome P4501A) monooxygenase enzyme system in cultured hepatocytes from Indian catfish, Heteropneustes fossilis (Bloch). We have quantified this induction by measuring the activity of the enzyme 7-ethoxyresorufin-O-deethylase (EROD), synthesized from CYP1A1 gene. The induction followed a dose-dependent relationship with carbofuran. The dose-dependent curve of EROD using carbofuran was very much similar with beta-napthoflavone, which is a known inducer of CYP1A1. Coexposure of these compounds to the culture media showed a synergistic effect on the enzyme activity. A blocker of aromatic hydrocarbon receptor, alpha-napthoflavone, blocked carbofuran-induced EROD activity in a dose-dependent manner. All these findings suggest that metabolism of carbofuran might be mediated by the CYP1A monooxygenase system through binding of the aromatic hydrocarbon receptor. We have also studied the superinduction phenomenon, which is a typical characteristic of the CYP1A gene in our system.     

Specific induction of hepatic cytochrome p4501a-2 in C57BL/6 and DBA/2 mice treated with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

The food mutagen/carcinogen amino-3-methylimidazo[4,5-f]quinoline (IQ) is activated by cytochrome p4501a-2 via N-hydrox-ylation; various P450s may contribute to detoxification via ring hydroxylation. Alterations in P450 levels by IQ treatment might therefore influence its toxicity. To examine the role of Ah locus genotype on the biochemical effects of IQ, C57BL/6 (Ah^bAh^b; p450Ia-½ inducible) and DBA/2 (Ah_dAh_d, noninducible) mice of both sexes were given IQ at varying doses, with different vehicles and routes of administration. Livers taken after 24 hours were assessed for total cytochrome p450 and activities of ethoxyresorufin-O-deethylase (EROD, a p4501a-l activity, inducible in Ah^b mice), meth-oxyresorufin-O-demethylase (MROD, a p4501a-2 activity), and benzyloxyresorufin-O-dealkylase (BzROD, an activity of p4502b). There was little effect on total cytochrome p450, but all three enzyme activities were often induced, a maximum of 2.5-fold, in both sexes and in DBA/2 as well as C57BL/6 mice. However, Western immunoblot analysis with monoclonal antibodies demonstrated an increase only in p4501a-2 protein. p4501a-l remained undetectable. A monoclonal antibody to p4502-b recognized one protein band in liver mi-crosomes from males and two bands in female mice of both strains. Amounts of these proteins were not altered by IQ treatment. Thus, IQ specifically, if moderately, induces its activating enzyme, p4501a-2, in a process that was not clearly related to Ah responsiveness.     

Possible implication of macrophages in the regulation of cytochrome P450 activities in carp (Cyprinus carpio)

Macrophages play a key role in the regulation of cytochrome P450 activity induced by immunostimulants in mammals. We investigated the effects of immunostimulants (LPS, dextran sulfate and tilorone) on biotransformation and macrophage activities in carp. The major effect of LPS was its capacity to inhibit 3-MC-induced cytochrome P450 activities in the liver and head kidney. Basal phase I activities were reduced by tilorone and dextran sulfate in immune organs. Tilorone and dextran sulfate differently modulated total cytochrome P450 contents and P4501A activities suggesting differential sensitivity for P450 classes. In immune organs, tilorone and dextran sulfate inhibited basal EROD activity. Tilorone inhibited 3-MC-induced EROD activity whereas dextran sulfate enhanced this activity. LPS and dextran sulfate increased ROS production by macrophages and all the immunostimulants induced macrophage activating factor (MAF) production. This study demonstrates for the first time in fish the capacity of CYP-regulated immunostimulants to activate macrophages and provides initial insight into the capacity of macrophages to regulate CYP activity induced by immunostimulants in fish.     

Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.     

Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes

Induction of cytochrome P4501A CYP1A in cultured cells can be used to determine the induction potencies of xenobiotics or complex environmental samples. This report describes the development of an enzyme linked immunosorbent assay ELISA for measurement of CYP1A expression in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes. Juvenile rainbow trout were injected with naphthoflavone BNF 25 mg kg-1 body weight to induce the synthesis of CYP1A. The CYP1A isoenzyme was purified, characterized by immunological cross reactivity and N terminal sequencing and used to prepare a monoclonal antibody in Balb C mice. The specificity of the antibody for CYP1A was proved by Western blotting of samples from control and BNF injected fish. Two ELISA methods, a direct and a competitive one, were evaluated, with both methods being of comparable sensitivity. Rainbow trout hepatocytes, maintained as monolayers in serum free, chemically defined medium, were exposed to naphthoflavone, and the induction response was measured both by 7 ethoxyresorufin O deethylase EROD activity and the direct ELISA method. Comparison between EROD activity and immunodetectable CYP1A protein can provide information on the catalytic efficiency of CYP1A.     

Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes

Induction of cytochrome P4501A CYP1A in cultured cells can be used to determine the induction potencies of xenobiotics or complex environmental samples. This report describes the development of an enzyme linked immunosorbent assay ELISA for measurement of CYP1A expression in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes. Juvenile rainbow trout were injected with naphthoflavone BNF 25 mg kg-1 body weight to induce the synthesis of CYP1A. The CYP1A isoenzyme was purified, characterized by immunological cross reactivity and N terminal sequencing and used to prepare a monoclonal antibody in Balb C mice. The specificity of the antibody for CYP1A was proved by Western blotting of samples from control and BNF injected fish. Two ELISA methods, a direct and a competitive one, were evaluated, with both methods being of comparable sensitivity. Rainbow trout hepatocytes, maintained as monolayers in serum free, chemically defined medium, were exposed to naphthoflavone, and the induction response was measured both by 7 ethoxyresorufin O deethylase EROD activity and the direct ELISA method. Comparison between EROD activity and immunodetectable CYP1A protein can provide information on the catalytic efficiency of CYP1A.     

Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N terminus and requires mitochondrial specific electron transfer proteins for activity

Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.     

Purification and Characterization of Cytochrome P450 Reductase from Liver Microsomes of Feral Leaping Mullet (Liza saliens)

NADPH-cytochrome P450 reductase was purified to electrophoretic homogeneity from detergent-solubilized liver microsomes from the leaping mullet (Liza saliens). The purified reductase was characterized with respect to spectral, electrophoretic, and biocatalytic properties. In addition, effects of pH, ionic strength, and the substrate concentration on the NADPH-dependent cytochrome c reductase activity of the purified fish liver cytochrome P450 reductase were studied. Cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 μmol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77,000 ± 1000 when electrophoresed on polyacrylamide gels under the denaturing conditions in the presence of SDS. The absorption spectrum of fish reductase showed two peaks at 378 and 455 nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4 and 7.8. The apparent K_m of the purified enzyme was found to be 7.69 μM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7, at room temperature, and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration was at or above the 70 μM. Furthermore, the purified enzyme was biocatalytically active in reconstituting the 7-ethoxyresorufin O-deethylase activity in the reconstituted system containing purified mullet liver cytochrome P4501A1 and lipid. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic, and biocatalytic properties. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 103–113, 1998     

Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics

The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed.     

Purified fusion enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 oxidoreductase

A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.     

Effects of polychlorinated biphenyls on cytochrome P450 induction in the chick embryo hepatocyte culture

The effects of pure synthetic polychlorinated biphenyl (PCB) congeners on the induction of cytochrome P450 and associated activities were examined in cultured chick embryo hepatocytes. Dose-response effects for the induction of total cytochrome P450 ethoxyresorufin-O-deethylase (EROD) activity, and benzphetamine demethylase (BPDM) activity were studied using 10 selected tetra- to hexachlorinated PCB congeners. These studies revealed that PCBs caused effects in the chick hepatocyte culture different from previously observed effects in rat liver. Based on their effects in chick hepatocytes, the PCBs could be categorized into two groups. The first group (consisting of 3,3',4,4'-PCB, 3,3',4,4',5-PCB, 3,3',4,4',5,5'-PCB, 2',3,3',4,5-PCB, 2,3,3',4,4',5'-PCB, and 2,3,4,4',5-PCB) induced total cytochrome P450 2.4- to 2.9-fold and EROD activity from 1-2 pmol/min/mg protein to 162-247. There was marked variation in potency, but all these congeners had a maximal inducing dose above which cytochrome P450 concentrations and EROD activities declined. BPDM activities were increased only slightly (1.2- to 1.6-fold) at the maximal cytochrome P450 inducing dose. The second group of congeners (consisting of 2,2',4,5,5'-PCB. 2,2',4,4',5,5'-PCB, and 2,2',3,4,4',6-PCB) induced total cytochrome P450 concentrations 4.0-fold and BPDM activities 2.2- to 2.6-fold with greatest activity occurring at the highest doses which could be added (10-50 microM). However, EROD activities were also increased by these congeners to 60-112 pmol/min/mg protein with declining activities seen at the highest PCB doses (i.e., resembling EROD induction patterns of the first group). The EROD induction patterns with these latter PCB congeners are noteworthy since these PCBs do not induce EROD activity in the rat. For both groups of PCB congeners, EROD induction was associated with increased accumulation of uroporphyrin in cultures exposed to exogenous 5-aminolevulinate. Studies investigating the reason for the depression of cytochrome P450 concentrations and/or EROD activities by high doses of the PCBs revealed that with the first group there was slightly decreased total protein synthesis, decreased total cell heme concentrations, and decreased accumulation of radiolabeled heme synthesized from 5-[14C]aminolevulinate. These changes might represent nonspecific toxic effects of the first group of PCBs. However, since these changes were not seen with the second group of PCBs, it is unlikely that either inhibition of heme synthesis or toxicity cause the depression of EROD activity with high PCB doses.     

Delayed gametogenesis and progesterone levels in soft-shell clams (Mya arenaria) in relation to in situ contamination to organotins and heavy metals in the St. Lawrence River (Canada)

Wild carp, Cyprinus carpio, were sampled in January and March 2000 in a section of the Anoia River (NE Spain) known to be polluted by estrogenic compounds. At each sampling time, three groups were distinguished: (1) apparently normal males; (2) apparently normal females; and (3) affected fish. The latter were characterized by the simultaneous development of male and female tissue in their gonads at a macroscopical level (six out of 31 fish sampled at this particular point), or testicular atrophy (three out of 31). Plasmatic and hepatic vitellogenin (VTG) levels and plasma testosterone (T) and estradiol (E2) were measured to observe the particular estrogenic response of the affected fish. Moreover, the response in the xenobiotic metabolizing capacity in liver was tested. This involved the analysis of mixed function oxygenase (MFO) system such as: total cytochrome P450 content, NAD(P)H cytochrome c reductases and the associated CYP1A1, EROD activity. Also, glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) as detoxifying enzymes were measured. Our results showed: (1) a highly variable VTG content in all fish groups; (2) an increase in sex hormones content in March for the female group; and (3) an enhanced xenobiotics metabolism in the affected fish group, measured as total cytochrome P450, EROD activity in the January survey and cytosolic GST in March. The observed increase in VTG, sex hormones and in most of the enzymatic activities from January to March that could also be attributed to higher water temperature.     

Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.     

Avian kidney mitochondrial hemeprotein P-4501 alpha: isolation, characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-4501 alpha from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-4501 alpha could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-4501 alpha traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57,000. The absolute spectrum of the P-4501 alpha (Fe3+) form gave a lambda max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501 alpha which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275-8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933-3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-4501 alpha catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3 beta-ol at the C-1 position exclusively with a turnover number of 0.03 min-1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.     

Purification and characterization of hepatic microsomal prostaglandin omega-hydroxylase cytochrome P-450 from pregnant rabbits

Prostaglandin omega-hydroxylase, designated as cytochrome P-450 LPG omega (P-450 LPG omega), has been purified, to a specific content of 15 nmol of cytochrome P-450/mg of protein, from liver microsomes of pregnant rabbits. The purified P-450 LPG omega was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and to have an apparent molecular weight of 52,000. The enzyme showed a maximum at 450 nm in the carbon monoxide (CO)-difference spectrum for its reduced form. This cytochrome P-450 efficiently catalyzed the omega-hydroxylation of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF 2 alpha), prostaglandin A1 (PGA1), and prostaglandin A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of myristate and palmitate, in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome b5. Various monovalent and divalent cations further stimulated these reactions in the presence of cytochrome b5. In addition, the reactions were also markedly enhanced by various organic solvents, such as ethanol and acetone. This cytochrome P-450 showed no detectable activity toward several xenobiotics tested. P-450 LPG omega was very similar or identical to the pulmonary prostaglandin omega-hydroxylase (P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., & Kusunose, M. (1984) J. Biochem. 96, 593-603) in its molecular weight, absorption spectra, catalytic activity, peptide mapping pattern, and N-terminal amino acid sequence. However, P-450 LPG omega was more unstable than P-450p-2 on storage. In sharp contrast to P-450p-2, P-450 LPG omega was not induced by progesterone.     

The role of cytochrome b 5 structural domains in interaction with cytochromes P450

To understand the role of the structural elements of cytochrome b ₅ in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b ₅ — microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b ₅ is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b ₅ to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b ₅ are mainly determined by the structure of the hemebinding domain.