Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.     

Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography

Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.     

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki approximately 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.     

Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.     

Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.     

Metabolism of the tricyclic antidepressant amitriptyline by cDNA-expressed human cytochrome P450 enzymes

The metabolism of amitriptyline was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6 and 2E1. CYP 2C19 was the most important enzyme with regard to the demethylation of amitriptyline, the quantitatively most important metabolic pathway. CYP 1A2, 3A4, 2C9 and CYP 2D6 also participated in the demethylation of amitriptyline. CYP 2D6 was the sole enzyme mediating the hydroxylation of amitriptyline, and (E)-10-OH-amitriptyline was exclusively produced. CYP 2E1 did not metabolize amitriptyline. Concerning the quantitative relations, CYP 2C19 and 2D6 exhibited high affinities with Km values in the range of 5-13 mumol/l, whereas the affinities of 1A2, 3A4 and 2C9 were somewhat lower with Km values ranging from 74 to 92 mumol/l. CYP 2C19 displayed the highest reaction capacity per mole with Vmax equal to 475 mol h-1 (mol CYP)-1. The other enzymes had Vmax values in the range of 90-145 mol h-1 (mol CYP)-1. Allowing for the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that, at therapeutic doses, on average about 60% of the metabolism depended on CYP 2C19. At toxic doses, CYP 2C19 is expected to be saturated, and CYP 3A4 may now play a dominant role in the metabolism.     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the initial acceptor in cytochrome c oxidase

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.     

Release of cytochrome c from liver mitochondria during permeability transition

The mitochondrial permeability transition (PT) follows opening of megachannels in the inner membrane and may be part of a programmed death pathway. Recently a role for cytochrome c in programmed cell death has been proposed, although its relationship to PT has not been been determined. We studied the release of cytochrome c from liver mitochondria undergoing PT. Well-coupled mitochondria treated with 5 mM atractyloside (ATR) or 100 microM calcium chloride were found to undergo PT and release cytochrome c into the incubation buffer within 5 minutes. Control mitochondria and mitochondria treated with the uncoupler FCCP did not undergo PT or release cytochrome c at 5 or 15 minutes. PT induced by ATR could be prevented by pretreatment with 10 microM cyclosporin A. Mitochondria incubated with ATR or calcium caused a 20-30% decrease in electron transfer rate via cytochrome c and cytochrome c oxidase. We conclude that cytochrome c release is an early event during mitochondrial PT, and is sufficient to decrease electron transfer through the terminal electron transport components of the mitochondrion.     

Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding

The mechanism of unfolding of ferricytochrome c induced by the surfactant sodium dodecyl sulfate has been studied by heme absorption, tryptophan fluorescence, circular dichroism, resonance Raman scattering, stopped-flow and time-resolved resonance energy transfer to obtain a comprehensive view of the whole process. Unfolding occurred at an almost specific molecular ratio of SDS/cytochrome c in the concentration range (20-50 microM) studied here. However there appears to be a point at approximately 0.6 mM SDS where unfolding begins to occur for lower cytochrome c concentrations. The kinetics of unfolding revealed only a single transition with a rate constant of 33 s(-1) (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of approximately 16 kJ/mol, indicating that other associated steps, if any, are too fast to be significantly populated. The free energy change (deltaG(o)) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS-unfolded cytochrome c shows only a partial decrease (25%), indicating that a significant amount of helical structure remains folded in contrast to a complete loss of helical structure in GdnHCl-denatured cytochrome c. The heme structure in SDS-unfolded cytochrome c, as deduced from heme absorption and resonance Raman spectra, shows a major population (approximately 95%) of mis-ligated histidine to the heme which acts as a kinetic trap in the folding process. The structural changes associated with cytochrome c unfolding were also monitored by time-resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in the native protein to 0.63 ns in the unfolded one, associated with a movement of Trp59 by 10 A away from heme. The maximum entropy method analysis of fluorescence decay indicated the growth of various conformational substates in SDS-unfolded cytochrome c in contrast to narrowly distributed conformations in the native protein. The refolding was comprised of three kinetic steps; the first was significantly fast (approximately 8 ms) and was assigned to the dissociation of His26 that paves the protein towards correct folding pathway. The other two slower steps probably arise from chain misorganization and prolyl isomerization. The absence of a burst-phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytochrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.     

Crystallization of a covalently-linked complex of adrenodoxin and adrenodoxin reductase

Two forms of mitochondrial adrenodoxin reductase from bovine adrenals and recombinant bovine adrenodoxin and adrenodoxin reductase expressed in Escherichia coli were isolated, purified to homogeneity and biochemically characterized. Recombinant adrenodoxin reductase was expressed as a single polypeptide; its retention time on DEAE-Fractogel coincides with the second form (F2) of the mitochondrial reductase. Two enzyme forms have similar adrenodoxin reductase activities in two types of systems comprising either cytochrome c or cytochrome P-450 (11 beta) as the terminal electron acceptor. Adrenodoxin and each of two reductase forms were cross-linked using 1-ethyl-3-(dimethyl-amino-propyl)carbodiimide. An effective two-step method for the purification of the active heterologous cross-linked complexes is suggested that enables purification of the functional complexes to homogeneity. The cross-linked bimolecular complex of adrenodoxin and adrenodoxin reductase was crystallized for the first time.     

Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stable than the reduced form, and 0.07 kcal mol(-1) is more stable than the Cyt c: azide complex at 25 degrees C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.     

Steroidogenic electron transport in adrenal cortex mitochondria

The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxin-adrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450scc to which this phospholipid binds.     

Electron transfer to cytochrome P-450scc limits cholesterol-side-chain-cleavage activity in the human placenta

The aim of this study was to determine whether electron transfer from adrenodoxin reductase and adrenodoxin limits the activity of cytochrome P-450scc in mitochondria from the human placenta. Mitochondria were disrupted by sonication to enable exogenous adrenodoxin and adrenodoxin reductase to deliver electrons to cytochrome P-450scc. After sonication, the rate of pregnenolone synthesis was greatly decreased relative to that by intact mitochondria, due to dilution of endogenous adrenodoxin and adrenodoxin reductase into the incubation medium. The addition of saturating concentrations of bovine or human adrenodoxin and bovine adrenodoxin reductase to the disrupted mitochondria gave an initial rate of pregnenolone synthesis that was 6.3-fold higher than that for intact mitochondria. Similar results were observed when 20alpha-hydroxycholesterol was used as substrate rather than endogenous cholesterol. The turnover number of cytochrome P-450scc in sonicated placental mitochondria supplemented with adrenodoxin and adrenodoxin reductase was comparable to that for the purified enzyme assayed under conditions where electron transfer was not limiting. Addition of exogenous adrenodoxin and adrenodoxin reductase to sonicated mitochondria from the pig corpus luteum and rat adrenal had a much smaller effect on pregnenolone synthesis compared with intact mitochondria, than observed for the placenta. We conclude that in the human placenta, electron transfer to cytochrome P-450scc is limiting, permitting pregnenolone synthesis to proceed at only 16% maximum velocity.     

Altering substrate specificity at the heme edge of cytochrome c peroxidase

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.     

Metabolism of clozapine by cDNA-expressed human cytochrome P450 enzymes

The metabolism of clozapine was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1. CYP1A2, 3A4, 2C9, 2C19, and 2D6 were able to N-demethylate clozapine. N-Oxide formation was exclusively catalyzed by CYP3A4. CYP2E1 did not metabolize clozapine. With regard to quantitative relationships, CYP1A2, 2C9, 2C19, and 2D6 displayed KM values ranging from 13 to 25 microM, whereas CYP3A4 had a 5-10 times higher KM value. CYP2C19 and 2D6 had the highest Vmax values (149-366 mol/hr/mol CYP). Taking into account the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that at therapeutic concentrations CYP2C19 and CYP3A4 each accounted for about 35% of the metabolism. At toxic concentrations, the relative importance of CYP3A4 increased.     

Virology on AIDS and AIDS dementia complex

Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P. M. Horowitz, and B. S. S. Masters (1995) Arch. Biochem. Biophys. 316, 267-274]. As in the case of CPR, FMN was relatively loosely bound to nNOS and the flavoprotein module, but FAD remained bound at concentrations of up to 2 M urea Protein fluorescence increased progressively with increasing urea concentration, but could not be correlated with changes in flavin binding. NADPH-cytochrome c reductase activity of both nNOS and the flavoprotein module, but not that of CPR, was stimulated at early time points by both urea and guanidine hydrochloride (GnHCl), with levels of initial activity returning to baseline values within 60 min after addition of the chaotropic agent. Thus, at 3-4 M urea, enhancements of reductase activities of 20- and 5-fold with nNOS and the flavoprotein module, respectively, were obtained. Comparable enhancements of 12- and 6- to 7-fold, respectively, were obtained with calmodulin (CaM)/ CaCl2 and 0.5 M GnHCl. Thus, the effects of urea and GnHCl mimicked the stimulating effects of CaM. Separate preincubations of nNOS and cytochrome c with urea or GnHCl prior to initiation of the reductase assay showed that sensitivity to chaotropic agent under these conditions was a property of nNOS and not of cytochrome c. Moreover, when the nonprotein electron acceptor 2,6-dichlorophenolindophenol was employed in place of cytochrome c, comparable stimulation of reductase activity was observed in the presence of either urea or GnHCl. Fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfate in the presence of either nNOS or the flavoprotein module was increased optimally between 3 and 4 M urea, consistent with simultaneous exposure of hydrophobic regions of both proteins to solvent and optimization of reductase activity. FMN release from nNOS, but not from the flavoprotein module, was enhanced by CaM. Addition of FMN or FMN + FAD to nNOS, in the presence or absence of urea, brought about a doubling of initial cytochrome c reductase activity, but did not prevent the eventual decline in activity to basal levels. These data are consistent with conformational changes which favor increased electron transfer similar to that achieved with nNOS in the presence of CaM.     

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.     

Combined effect of cadmium and nickel on rat hepatic monooxygenases: possible stimulation of certain cytochrome P-450 isozymes

When male rats were given either a single dose of cadmium (3.58 mg CdCl2.6H2O/kg, i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl2.6H2O/kg, s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg/kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg/kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.     

Effects of temperature on the kinetics of the gated electron-transfer reaction between zinc cytochrome c and plastocyanin. Analysis of configurational fluctuation of the diprotein complex

This is a study of the effects of temperature (in the range 273.3-307.7 K) and of ionic strength (in the range 2.5-100 mM) on the kinetics of photoinduced electron-transfer reaction 3Zncyt/pc(II)--> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and cupriplastocyanin at pH 7.0. In order to separate direct and indirect effects of temperature on the rate constants, viscosity of the solutions was fixed, at different values, by additions of sucrose. The activation parameters for the reaction within the preformed complex, at the low ionic strength, are delta H++ = 13 +/- 2 kJ/mol and delta S++ = -97 +/- 4 J/K mol. The activation parameters for the reaction within the encounter complex, at the higher ionic strength, are delta H++ = 13 +/- 1 kJ/mol and delta S++ = -96 +/- 3 J/K mol. Evidently, the two complexes are the same. The proteins associate similarly in the persistent and the transient complex, i.e., at different ionic strengths. In both complexes, however, electron transfer is gated by a rearrangement, as previous studies from this laboratory showed. Changes in the solution viscosity modulate this rearrangement by affecting delta H++, not delta S++. The activation parameters are analyzed by empirical methods. The thermodynamic parameters delta H and delta S for the formation of the complex Zncyt/pc(II) are determined and related to changes in hydrophilic and hydrophobic surfaces upon protein association in three configurations. A difference between the values of delta H for the configuration providing optimal electronic coupling between the redox sites and the configuration providing optimal docking equals the experimental value delta H++ = 13 kJ/mol for the rearrangement of the latter configuration into the former. Enthalpy of activation may reflect a change in the character of the exposed surface as the diprotein complex rearranges. Entropy of activation may reflect tightening of the contact between the associated proteins.