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PubMed1989

Distribution and induction of cytochrome P-450 and two cytochrome P-450-dependent monooxygenase activities in rat liver parenchymal cell subpopulations separated by centrifugal elutriation.

Liver parenchymal cells from the periportal and centrilobular zones differ in their morphological, biochemical and functional characteristics. In an effort to obtain fractions enriched in either periportal or centrilobular cells, isolated rat liver parenchymal cells were separated into five subpopulations by centrifugal elutriation. The mean diameters of the cells present in fractions I-V were 19.6, 21.1, 21.8, 22.7 and 23.5 micron, respectively. The content of cytochrome P-450 as well as benzphetamine N-demethylase and 7-ethoxyresorufin O-deethylase activities were higher in the larger parenchymal cells than in the smaller ones. After administration of phenobarbital the content of cytochrome P-450 was approximately two-fold greater in the cells present in fractions 3-5, when compared to the same subpopulations isolated from untreated rats; the activity of benzphetamine N-demethylase was enhanced to a similar extent in all five fractions. 3-Methylcholanthrene treatment resulted in a significant increase of cytochrome P-450 content and 7-ethoxyresorufin O-deethylase activity in all five fractions: both parameters were slightly higher in fractions 4 and 5 than in fractions 1 and 2. In conclusion, the elutriated liver parenchymal cells seem to preserve the biochemical heterogeneity observed in the intact liver; the potential enrichment of periportal and centrilobular cells in the different fractions by centrifugal elutriation is discussed.

Alanine Transaminase↗
PubMed1988

Formation of electrostatically-stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase.

Electron transfer from yeast ferrous cytochrome c to H2O2-oxidized yeast cytochrome c peroxidase has been studied using flash photoreduction methods. At low ionic strength (mu less than 10 mM), where a strong complex is formed between cytochrome c and peroxidase, electron transfer occurs rather slowly (k approximately 200s-1). However, at high ionic strength where the electrostatic complex is largely dissociated, the observed first-order rate constant for peroxidase reduction increases significantly reaching a concentration independent limit of k approximately 1500 s-1. Thus, at least in some cases, formation of an electrostatically-stabilized complex can actually impede electron transfer between proteins.

Cytochrome c Group↗
PubMed1990

Protein conformational "annealing": binding to cytochrome c refolds the active site region of recombinant cytochrome c peroxidase.

When initially isolated with heme reconstitution, recombinant cytochrome c peroxidase molecules exhibit a conformation, revealed by visible spectra which observably differ from the corresponding holo proteins isolated from yeast. Binding yeast iso-1 cytochrome c to these recombinant cytochrome c peroxidases (either in solution or via an affinity column) catalyses a local refolding of the recombinant proteins to a form that is indistinguishable from the native (yeast) protein.

Binding Sites↗
PubMed1990

Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels.

A double mutant of Saccharomyces cerevisiae, in which CYCL gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild-type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc1 complex is dispensable for yeast mitochondrial respiration in cells with the wild-type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol- phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.

Blotting, Southern↗
PubMed1992

Phosphorylation of cytochrome b6 by the LHC II kinase associated with the cytochrome complex.

The cytochrome b6 polypeptide present in cytochrome b6/f preparations from spinach thylakoids is phosphorylated concomitantly with the autophosphorylation of the 64 kDa polypeptide identified as the redox-controlled LHCII kinase. The N-terminal sequence of the 64 kDa kinase and sequence analysis of cytochrome b6 indicate the existence of putative phosphorylation sites in both proteins.

Amino Acid Sequence↗
PubMed2005

Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase.

The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.

Animals↗
PubMed2005

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro.

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.

Androgens↗
PubMed1986

The role of cytochrome a in the proton pump of cytochrome-c oxidase.

Cytochrome-c oxidase of bovine heart mitochondria was depleted of copper A by dialysis against 1 M KCN in the presence of dodecylmaltoside. There was no difference of the pH-dependence of the midpoint potential between the intact and the copper-depleted enzyme. Oxidation of reduced cytochrome a2+a3(3+).CN complex released about 1 proton/electron in the medium at pH 7.6. This release was inhibited by N,N'-dicyclohexylcarbodiimide. Again there was no difference between the intact and Cu-depleted enzyme. This limits the role of copper A in the mechanism of the proton pump. On the other hand, these experiments showed that cytochrome a could be a component of the proton pump.

Animals↗
PubMed2000

Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551.

The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.

Amino Acid Substitution↗
PubMed1993

Assembly of the Rieske iron-sulfur subunit of the cytochrome bc1 complex in the Escherichia coli and Rhodobacter sphaeroides membranes independent of the cytochrome b and c1 subunits.

The Rieske iron-sulfur subunit of the cytochrome bc1 complex from Rhodobacter sphaeroides has been expressed in Escherichia coli and also in a strain of Rb. sphaeroides lacking the other subunits of the bc1 complex. PCR products encoding the full-length subunit were introduced into expression vectors to produce the subunit alone or the subunit fused behind the mature portion of the E. coli maltose binding protein (MBP), but lacking the MBP signal sequence. These proteins are both located in the cytoplasmic membrane. The unfused Rieske subunit assembles a Rieske-like iron-sulfur cluster, but with EPR characteristics which differ from the normal rhombic signal observed in the cytochrome bc1 complex. The overproduced MBP fusion protein, on the other hand, does not contain an EPR-detectable iron-sulfur cluster. Subfragments of the Rieske subunit lacking the amino-terminal hydrophobic anchor also lack the iron-sulfur cluster were expressed in E. coli. When expressed in Rb. sphaeroides in the absence of the cytochrome b and c1 subunits, the fully metalated Rieske subunit with the diagnostic gy = 1.90 EPR signal is observed in the cytoplasmic membrane. The fact that the Rieske subunit has an assembled iron-sulfur cluster and is bound to either the E. coli or the Rb. sphaeroides membrane in the absence of the other subunits of the bc1 complex demonstrates a mode of membrane attachment independent of the other components of the complex. These data are consistent with models in which the Rieske subunit is bound to the membrane via a single membrane-spanning helix located near the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1994

Reaction of horse cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I.

The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a number of CcP(MI) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-191 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 x 10(8) M-1 s-1. Ferrocytochrome c then reduces the oxyferryl heme Fe(IV) in CMPII with a rate constant of 2.0 x 10(6) M-1 s-1. The rate constant for the reduction of the radical is nearly independent of pH from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(IV) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of the electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(MI). The intracomplex rate constants for reduction of the radical in these mutants by the ruthenium-labeled cytochrome c derivatives are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Internal electron transfer in cytochrome c oxidase: evidence for a rapid equilibrium between cytochrome a and the bimetallic site.

Internal electron-transfer reactions in cytochrome oxidase following flash photolysis of the CO compounds of the enzyme reduced to different degrees (2-4 electron equiv) have been followed at 445, 605, and 830 nm. Apart from CO dissociation and recombination, two kinetic phases are seen both at 445 and at 605 nm with rate constants of 2 x 10(5) and 1.3 x 10(4) s-1, respectively; at 605 nm, an additional phase with a rate constant of 400 s-1 is resolved. At 830 nm, only the second reaction phase (rate constant of 1.3 x 10(4) s-1) is observed. The amplitude of the first phase is largest with the two-electron-reduced enzyme, whereas that of the second phase is maximal at the three-electron-reduction level. Neither phase shows any marked pH dependence. The reaction in the first phase has a free energy of activation of 41 kJ mol-1 and an entropy of activation of -14 JK-1 mol-1. Analysis suggests that the two rapid reaction phases represent internal electron redistributions between the bimetallic site and cytochrome a, and between cytochrome a and CuA, respectively. The slow phase (400 s-1) probably involves a structural rearrangement.

Animals↗
PubMed1988

Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity.

A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.

Amino Acid Sequence↗
PubMed1990

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin.

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.

Cytochrome c Group↗
PubMed1977

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.

Anaerobiosis↗
PubMed2002

Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo(3) from Escherichia coli.

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.

Animals↗
PubMed2002

Crowding by trisaccharides and the 2:1 cytochrome c-cytochrome c peroxidase complex.

Proteins are designed to function under crowded conditions where the solute concentration can reach 400 g/L, but they are almost always studied in dilute solutions. To address this discrepancy, we have undertaken a series of studies to determine the effects of high solute concentrations on the thermodynamics of protein equilibria. Recently, we used isothermal titration calorimetry (ITC) to show that high concentrations of mono-, di-, and tetrasaccharides have a small stabilizing effect on the crystallographically defined cytochrome c binding site on yeast ferricytochrome c peroxidase [Morar, A. S., Wang, X., and Pielak, G. J. (2001) Biochemistry 40, 281-285]. Here, we use this technique to show that trisaccharides increase the apparent thermodynamic binding constants for both cytochrome c binding sites on the peroxidase. Mutagenesis studies confirm that the second site includes Asp 148 on the peroxidase. Binding of both cytochrome c molecules is exothermic. The data are interpreted by assuming either the presence or absence of intersite interactions.

Aspartic Acid↗
PubMed1996

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase.

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.

2,2'-Dipyridyl↗