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PubMed2005

Bimolecular fluorescence complementation analysis of cytochrome p450 2c2, 2e1, and NADPH-cytochrome p450 reductase molecular interactions in living cells.

Interactions between cytochromes P450 (P450s) and P450 reductase are required for enzymatic activity, and homo- or heterooligomerization of P450s may also be functionally important. Bimolecular fluorescence complementation (BiFC) was used to examine P450 interactions in a natural membrane context within living cells. BiFC detects protein interactions in living cells by reconstitution of a fluorescent protein from two fragments that are fused to the two interacting proteins. Nonspecific protein-protein interactions were detected if proteins were expressed at high levels. At low protein expression levels, homo-oligomerization of P450 2C2, but not P450 2E1, and interactions of these P450s with P450 reductase were detected by BiFC, consistent with interactions detected previously by fluorescence resonance emission transfer. Weak interaction of P450 2C2 with P450 2E1 and homooligomerization of P450 reductase was also detected by BiFC. Homo-oligomerization of the N-terminal P450 2C1 signal anchor sequence and interactions between the signal anchor and full-length P450 2C2 were detected, suggesting that homo-oligomerization of P450 2C2 is mediated by the signal anchor. However, interactions between the signal anchor and either P450 2E1 or P450 reductase were not detected by BiFC. Although high concentrations of the substrate lauric acid increased BiFC for both P450 2E1 and P450 2C2 with P450 reductase, the concentration dependence did not correlate with reported K(m) values. These results demonstrate that BiFC is an effective method to study the complex protein interactions that occur within the microsomal P450 system in living cells.

Animals↗
PubMed2004

Novel reversible inactivation of cytochrome P450 2E1 T303A by tert-butyl acetylene: the role of threonine 303 in proton delivery to the active site of cytochrome P450 2E1.

This report investigates and characterizes the mechanism for the novel reversible inactivation of a T303A mutant of rabbit cytochrome P450 (P450) 2E1 by tert-butyl acetylene (tBA). P450 2E1 T303A was inactivated in a time-, concentration-, and NADPH-dependent manner through the formation of two tBA adducts to the P450 heme. Interestingly, losses in enzymatic activity and in the reduced CO spectrum of the tBA-inactivated T303A mutant could be restored to the samples after an overnight incubation at 4 degrees C. Removal of free tBA and NADPH from the tBA-inactivated T303A samples by spin column gel filtration demonstrated that the observed reversibility was time-dependent and was not significantly affected by the presence or absence of NADPH or tBA. Furthermore, the recovery of native heme was dependent on the native P450 enzyme structure. Electrospray ionization liquid chromatography-tandem mass spectrometry analysis under nondenaturing conditions of a preacidified tBA-inactivated T303A sample yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns characteristic of a tBA adduct to the P450 heme. These adducts were absent in nonacidified samples subjected to the same conditions. In contrast, tandem mass spectrometry analysis of both non- and preacidified tBA-inactivated wild-type 2E1 samples yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns similar to the preacidified T303A mutant adducts. These results lend insight into the reversible inactivation mechanism of the tBA-inactivated T303A mutant and suggest a role for the highly conserved threonine 303 residue in proton donation to the P450 2E1 active site and the stabilization of a reactive intermediate during substrate metabolism by P450.

Acetylene↗
PubMed1996

Characterization of six in vitro reactions mediated by human cytochrome P450: application to the testing of cytochrome P450-directed antibodies.

The identification of cytochrome P450 (CYP) isozymes mediating metabolic pathways of drugs has become increasingly important in anticipating pharmacokinetic drug interactions. The activity of individual CYPs can be monitored in vitro with human liver microsomes by means by relatively specific metabolic reactions: for CYP1A1/2, phenacetin O-deethylation; for CYP2C9, phenytoin 4-hydroxylation; for CYP2C19, S-mephenytoin 4'-hydroxylation; for CYP2D6, dextromethorphan O-demethylation; for CYP3A3/4, alprazolam 4-hydroxylation, and for CYP2E1, chlorzoxazone 6-hydroxylation. We determined the kinetic parameters (Vmax, Km) of these reactions and utilized them to test a monoclonal rat liver CYP1A1 antibody, a monoclonal rat liver CYP2E1 antibody, a polyclonal rabbit anti-rat liver CYP3A1 antibody, and a polyclonal goat anti-rat liver CYP2C11 antibody for their specificity and inhibitory capacity. The CYP1A1 monoclonal antibody (MAb), the CYP2E1 MAb, and the CYP3A1 polyclonal antibody (PAb) inhibited only their respective index reactions. The CYP2C11 PAb inhibited both phenytoin 4-hydroxylation and S-mephenytoin 4'-hydroxylation. At a microsomal versus antibody protein mass ratio of 1:15, 4-hydroxyalprazolam formation was reduced by 73.4% of control with the CYP3A1 PAb; 4'-hydroxymephenytoin formation decreased by 66.3% and 4-hydroxyphenytoin formation by 43.4% with the CYP2C11 PAb; phenacetin O-deethylation was reduced by 39.7% with the CYP1A1 MAb, and 6-hydroxychlorzoxazone formation decreased by 30.0% with the CYP2E1 MAb. Thus, all antibodies tested are at least CYP subfamily specific. The PAbs exhibited greater than 60% inhibition versus the CYP3A3/4- and the CYP2C19-mediated reactions, whereas the MAbs produced less than 50% inhibition for their respective index reactions. Because of their limited inhibitory capacity, MAbs may be correspondingly limited as tools for identification of human CYP enzymes via immunoinhibition studies.

Animals↗
PubMed1995

Drug interactions and the cytochrome P450 system. The role of cytochrome P450 2C19.

On the basis of the data currently available, the wild type cytochrome P450 2C19 (CYP2C19) gene appears to be absent in 2 to 6% of Caucasian populations and up to 20% of Asian populations. A poor metaboliser phenotype therefore results. The CYP2C19 phenotype can be determined clinically by use of either mephenytoin or omeprazole as the probe. Since both drugs are metabolised primarily by CYP2C19 in human liver microsomes, they are both useful, competitive inhibitors of CYP2C19 activity available to researchers working in vitro. The 2 known mutant alleles of CYP2C19 do not result in the expression of active enzyme and can be tested by using small samples of whole blood. No rapid mutants have been identified. A number of drugs have been shown to inhibit CYP2C19 in vivo, including fluvoxamine and fluoxetine. Important drug interactions may result from inhibition of hepatic CYP2C19 activity in extensive metabolisers or from the interaction of CYP2C19 substrates with other pathways in poor metabolisers.

Animals↗
PubMed2005

Evaluation of data in terms of two-dimensional random walk model: interaction between NADH-cytochrome b5 reductase and cytochrome b5.

Normally, bimolecular reactions are analyzed in terms of the Smoluchowski theory. However, when one attempts to generalize this analysis to cases where diffusion proceeds in two other than in three dimensions, one soon encounters severe conceptual difficulties. Although kinetic studies of membrane enzymes are generally difficult because the usual kinetic formalism refers to nonaggregated homogenous solutions, a major goal of our research is to define the molecular mechanism(s) by which alterations in membrane-bound substrate contents affect the enzyme activity in the same membrane. For that purpose, a simplified random-walk model was adopted in the present work. The enzyme reaction in the two-dimensional membrane could be calculated theoretically by applying the classical analysis of heat equation. As a result, the theoretical rate equation well accounting experimental findings was derived on the model of the liver microsomal NADH-cytochrome b5 reductase reaction. Furthermore, it was found that the modification of the simple rigid-sphere collision theory by including a term called the steric factor was not necessary in this derived equation.

Animals↗
PubMed1992

Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene.

1. A polyclonal, monospecific antibody to a constitutive, diabetes-inducible and insulin-reversible cytochrome P-450 isozyme (RLM6) was used to screen a male rat liver cDNA library in lambda gt 11. Six clones harbouring the RLM6 cDNA insert were isolated initially from the expression library and three of these were further plaque-purified and sub-cloned. A 1.1 Kb cDNA insert, representing approximately 65% of the expected full length cDNA was characterized by restriction endonuclease mapping and sequenced by the dideoxy chain-termination method. Comparison of the nucleotide sequence of RLM6 cDNA to that of ethanol-inducible P4502E1 rat cDNA showed the two cDNAs to be identical, the RLM6 cDNA corresponding to nucleotides 310-1402 of the P4502E1 sequence. 2. RLM6 cDNA probe was used in Northern blot and RNA dot blot hybridization analysis to demonstrate that both streptozotocin-induced diabetes and fasting significantly elevated the steady-state level of RLM6 mRNA in male rat liver. Increased RLM6 mRNA level in the diabetic rat resulted in increased RLM6 apoprotein synthesis when polysomal RNA was used in a cell-free, protein-synthesizing system, indicating that the elevated RLM6 level observed in diabetic rats was correlated directly with the increased RLM6 mRNA concentration. 3. Daily insulin treatment of diabetic rats reversed the diabetes-dependent increase in RLM6 mRNA in a time-dependent manner, returning to control values after approximately 2 weeks of continuous insulin treatment. This insulin-dependent decrease of the RLM6 mRNA level was paralleled by a similar time-dependent decrease in serum acetone concentration. 4. Treatment of the male diabetic rat with testosterone also resulted in a decrease in both RLM6 mRNA and in vitro translated apoprotein. 5. Modulation of RLM6 mRNA level in the diabetic rat by insulin and testosterone, and the nucleotide sequence similarity with that of P4502E1 confirms that diabetes-inducible P450RLM6 and ethanol-inducible P4502E1 are coded for by the same gene.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence-based prevention (EBP): approach to lung cancer prevention based on cytochrome 1A1 and cytochrome 2E1 polymorphism.

Now that the human genome has been sequenced, we consider that combinations of 3 to 10 million polymorphic loci scattered throughout the genome contribute to individual differences. Genetic polymorphism analysis is useful for the made-to-order medical treatment of patients and the prevention of disease onset in normal healthy individuals. Individually-tailored disease prevention based on findings such as genetic polymorphism analysis results can be grasped by the concept of evidence-based prevention (EBP). In this paper we outline cytochrome P450 (CYP) 1A1 and 2E1 polymorphism-related differences in metabolic activation of carcinogens in relation to the risk of lung cancer, and explain single nucleotide polymorphism (SNP). We also compare the reports to date on SNP types in CYP1A1 and CYP2E1 in relation to the risk of lung cancer with our study results, examine survival rates of lung cancer patients by CYP1A1 or CYP2E1 genotype and discuss the applicability of SNP research to EBP.

Cytochrome P-450 CYP1A1↗
PubMed1991

Acidic cytochrome c6 of unicellular cyanobacteria is an indispensable and kinetically competent electron donor to cytochrome oxidase in plasma and thylakoid membranes.

Cytochromes c6 from three cyanobacteria were tested as substrates for membranous cyt. c oxidase(aa3) of Anacystis and Synechocystis using intact spheroplasts or isolated plasma(CM) and thylakoid(ICM) membranes. Neither spheroplasts nor CM/ICM gave significant O2 uptake rates with NADH without added cyt. c. Horse cyt. c (at low ionic strength) or cyt. c6 from Anacystis, Synechocystis or Microcystis (at high ionic strength) supported substantial HCN- & CO-sensitive NADH oxidase activity, consistent with in vivo O2 uptake. Cyanobacterial respiratory electron transport involves NADH dehydrogenase(fpN), plastoquinone, cyt. b/c(f), cyt. c6 & cyt. aa3, in both CM & ICM. In ICM, fpN and cyt. aa3 are functionally replaced in the light by PS II and PS I, respectively. In both membranes, cyt. c6 is an obligatory electron donor to cyt. aa3 &/or to P700. Respiratory action of acidic cyt. c6 (in unicellular species) may be unmasked only under conditions of elevated ionic strength.

Animals↗
PubMed1980

[Parameters influencing inactivation-dissociation/reactivation-association phenomena induced by variations of ionic strength of L (+)lactate: cytochrome c oxidoreductase (cytochrome b2) extract of the yeast Hansenula anomala].

H-flavocytochrome b2, a tetramer enzyme, is inactivated, at low ionic strength and can be reactivated, increasing the ionic strength of the medium. The inactivation-reactivation process was structurally manifested by a dissociation-association phenomenon between subunits. It was clearly shown that the inactivation-dissociation process appeared independent of enzyme concentration whereas the reactivation-association phenomenon was enzyme concentration dependent. However, proteins protect H-flavocytochrome b2 from inactivation-dissociation, only when electrostatic interactions are possible between the two proteins: Horse heart cytochrome c was a good protector whereas serum albumin had no protector effect.

Animals↗
PubMed1984

Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme.

Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.

Aging↗
PubMed1996

Identification of human cytochrome P450 isoforms involved in the 3-hydroxylation of quinine by human live microsomes and nine recombinant human cytochromes P450.

Studies using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie-Hofstee plots for the formation of 3-hydroxyquinine exhibited apparently monophasic behavior for all of the 10 different microsomal samples studies. There was interindividual variability in the kinetic parameters, as follows: 1.8-, 3.2- and 3.5-fold for K(m) Vmax and Vmax/K(m), respectively. The mean +/- S.D. values for K(m), Vmax and Vmax/K(m) were 106.1 +/- 19.3 microM, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 microliters/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine and the metabolic activities for substrates of the respective CYP isoforms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testosterone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4'-hydroxylase activities was also observed. However, no significant correlation existed between the 3-hydroxylation of quinine and the oxidative activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and troleandomycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinine by human liver microsomes with respective mean IC50 values of 0.026 microM and 28.9 microM. Anti-CYP3A antibodies strongly inhibited quinine 3-hydroxylation, whereas weak inhibition was observed in the presence of S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to the 3-hydroxylation of quinine, compared with the minor activity of CYP2C19 and little discernible or no effect of other CYP isoforms. Collectively, these data suggest that the 3-hydroxylation of quinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance in this major pathway of quinine in humans.

Antibodies↗
PubMed2000

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.

Acetyltransferases↗
PubMed1999

Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

Aerobiosis↗
PubMed2001

Role of LYS271 and LYS279 residues in the interaction of cytochrome P4501A1 with NADPH-cytochrome P450 reductase.

It has been proposed that negatively charged amino acids on the surface of reductase and positively charged amino acids on the surface of P450 mediate the binding of both proteins through electrostatic interactions. In this study, we used a site-directed mutagenesis approach to determine a role for two lysine residues (Lys271 and Lys279) of cytochrome P4501A1 in the interaction of P4501A1 with reductase. We prepared two mutants P4501A1Ile271 and P4501A1Ile279 with a mutation of the lysine at positions 271 and 279, respectively. We observed a strong inhibition (>80%) of the 7-ethoxycoumarin and ethoxyresorufin deethylation activity in the reductase-supported system for both mutants. In the cumene hydroperoxide-supported system, P4501A1Ile279 exhibited wild-type activity, but the P4501A1Ile271 mutant activity remained low. The CD spectrum and substrate-binding assay indicated that the secondary structure of P4501A1Ile271 is perturbed. To evaluate further the involvement of these P4501A1 lysine residues in reductase binding, we measured the KM of reductase for wild type and mutants. Both wild type and P4501A1Ile271 reached saturation in the range of reductase concentrations tested with KM values 5.1 and 11.2 pM, respectively. The calculated KM value for P4501A1Ile279 increased 9-fold, 44.4 pM, suggesting that the mutation affected binding of reductase to P4501A1. Stopped-flow spectroscopy was employed to evaluate the effect of mutations on electron transfer from reductase to heme iron. Both wild type and P450Ile279 showed biphasic kinetics with a approximately 40% participation of the fast step in the total activity. On the other hand, only single-phase kinetics for iron reduction was observed for P450Ile271, suggesting that the low activity of this mutant can be attributed not only to major structural changes but also to a disturbance in the electron transport.

Amino Acid Substitution↗
PubMed2001

The N-terminus of the Qcr7 protein of the cytochrome bc(1) complex in S. cerevisiae may be involved in facilitating stability of the subcomplex with the Qcr8 protein and cytochrome b.

Subunit 7 or the Qcr7 protein of the cytochrome bc(1) complex in yeast is a nuclear-encoded 14-kDa protein and is essential for formation of a functional bc(1) complex and respiration. It was shown previously that the N-terminal region of the Qcr7 protein might play little role in import but may be essential for correct assembly of the bc(1) complex. To examine the role of the N-terminus in assembly of the bc(1) complex, various N-terminus deletion and point mutants of the QCR7 gene were expressed in yeast strains where the endogenous QCR7 gene has been inactivated. Deletion of the first 8-15 residues after methionine at the N-terminus of the Qcr7 protein was studied and it was shown that except for the Qcr7p-Delta7 mutant, the strains overexpressing deletion mutants (Qcr7p-Delta8 to Qcr7p-Delta14) displayed decreased steady-state levels of iron-sulfur protein (ISP) and 14-kDa (Qcr7) and 11-kDa (Qcr8) subunits, as well as a respiratory defect. It was shown that introducing mutations at the N-terminus of the QCR7 gene in the Delta7 context had more drastic effects on respiration and assembly than in the full-length context, suggesting that the first seven residues at the N-terminus have a role in increasing the stability of the holocomplex. This led to a respiratory-deficient phenotype and drastic decrease in the steady-state levels of Qcr7 (14-kDa) and Qcr8 (11-kDa) proteins. The mutants Qcr7p-Delta7 (R10I), Qcr7p-Delta7 (G12V), Qcr7p-Delta7 (D13G), Qcr7p-Delta7 (D13V), and Qcr7p-Delta7 (D13Y) showed decreases in the steady-state levels of ISP and 14- and 11-kDa subunits, as well as defects in respiration. These results are interpreted in the light of the X-ray crystal structure of the yeast bc(1) complex.

Cytochrome b Group↗
PubMed1995

Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

Binding Sites↗
PubMed1998

Structural basis for the binding of an anti-cytochrome c antibody to its antigen: crystal structures of FabE8-cytochrome c complex to 1.8 A resolution and FabE8 to 2.26 A resolution.

A complete understanding of antibody-antigen association and specificity requires the stereochemical description of both antigen and antibody before and upon complex formation. The structural mechanism involved in the binding of the IgG1 monoclonal antibody E8 to its highly charged protein antigen horse cytochrome c (cyt c) is revealed by crystallographic structures of the antigen-binding fragment (Fab) of E8 bound to cyt c (FabE8-cytc), determined to 1.8 A resolution, and of uncomplexed Fab E8 (FabE8), determined to 2.26 A resolution. E8 antibody binds to three major discontiguous segments (33 to 39; 56 to 66; 96 to 104), and two minor sites on cyt c opposite to the exposed haem edge. Crystallographic definition of the E8 epitope complements and extends biochemical mapping and two-dimensional nuclear magnetic resonance with hydrogen-deuterium exchange studies. These combined results demonstrate that antibody-induced stabilization of secondary structural elements within the antigen can propagate locally to adjacent residues outside the epitope. Pre-existing shape complementarity at the FabE8-cytc interface is enhanced by 48 bound water molecules, and by local movements of up to 4.2 A for E8 antibody and 8.9 A for cyt c. Glu62, Asn103 and the C-terminal Glu104 of cyt c adjust to fit the pre-formed VL "hill" and VH "valley" shape of the grooved E8 paratope. All six E8 complementarity determining regions (CDRs) contact the antigen, with CDR L1 forming 46% of the total atomic contacts, and CDRs L1 (29%) and H3 (20%) contributing the highest percentage of the total surface area of E8 buried by cyt c (550 A2). The E8 antibody covers 534 A2 of the cyt c surface. The formation of five ion pairs between E8 and flexible cyt c residues Lys60, Glu62 and Glu104 suggests the importance of mobile regions and electrostatic interactions in providing the exquisite specificity needed for recognition of this extremely conserved protein antigen. The highly homologous VL domains of E8 and anti-lysozyme antibody D1. 3 achieve their distinct antigen-binding specificities by expanding the impact of their limited sequence differences through the recruitment of different sets of conserved residues and distinctly different CDR L3 conformations.

Animals↗