Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems

A series of equilibrium and kinetic measurements involving the oxidation-reduction properties of purified rat liver NADPH-cytochrome P-450 reductase and eight different purified rat liver cytochromes P-450 (P-450s) were carried out. Apparent spin states of P-450 iron were determined in the absence and presence of a number of known substrates by using second-derivative and conventional near-UV absorbance spectroscopy. Many of the substrates examined did not produce significant changes in the apparent iron spin state, even when binding could be demonstrated with equilibrium dialysis. Further, the spin state was not correlated to catalytic activity of the P-450s in reconstituted enzyme systems. The oxidation-reduction potentials were determined for the ferric/ferrous couples of each of the eight P-450s in the presence and absence of known substrates, as well as other proteins suspected of altering the potentials. The midpoint potential (Em,7) ranged from -350 to -289 mV for the P-450s under these conditions. In some cases Em,7 was raised with the addition of substrates, but the extent of the increase was no greater than +33 mV. The Em,7 of one P-450 (P-450 beta NF/ISF-G) was not changed significantly when the fraction of high-spin iron varied between 11 and 67%. Steady-state spectral studies provided evidence for the accumulation of an oxygenated ferrous intermediate (or a derived product) of one P-450 (P-450PB-B) in the presence of a substrate, cyclohexane. Studies on the donation of electrons from cytochrome b5 and a series of dyes to this complex suggest that it has an effective Em,7 (for reduction) of approximately +50 mV. In studies with one of the P-450s, steady-state spectral studies indicated that the three-electron-reduced form of NADPH-P-450 reductase accumulates, consistent with the view that this form of the reductase is involved in the reduction of P-450 from the ferric to the ferrous state.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

Substrate specificity of the carbon monoxide-dependent cytochrome P-450 kinetics

The substrate-dependent kinetics of the carbon monoxide-inhibited cytochrome P-450 activity and its light reversibility is reinvestigated in microsomal preparations. In order to find out whether the substrate specificity is mediated by an isoenzyme-specific binding of carbon monoxide with different dissociation constants an experimental design has been chosen where it could be established that essentially the same isoenzyme component was involved in two different monooxygenase reactions, i.e., the O-dealkylation of 7-ethoxycoumarin and the 7-hydroxylation of coumarin. The dissociation constant kD(CO) of the ferrous cytochrome P-450 carbon monoxide complex is 6-fold higher in the presence of 7-ethoxycoumarin than in the presence of coumarin. But the light-induced relative changes of the Warburg partition coefficient for the 7-ethoxycoumarin deethylation and for coumarin 7-hydroxylation do not differ remarkably from each other. These relative changes are shown to represent the ratio of the photoinduced rate constant to the spontaneous rate constant of the dissociation for the ferrous cytochrome P-450 carbon monoxide complex. The differences in the dissociation constants are assigned to substrate specific effects on the carbon monoxide binding, indicating a substrate-specific change of the free binding enthalpy for carbon monoxide.     

Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Chemical reactivities of bleomycin

Ferric bleomycin was tested for its ability to catalyze a set of six oxidative reactions characteristic of the heme-containing proteins, cytochrome P-450 and chloroperoxidase. These reactions included peroxyacid decarboxylation and aliphatic hydroxylation as typical cytochrome P-450 chemistries. Peroxyacid-supported oxygen evolution and hydrogen peroxide-mediated chlorination were utilized as characteristic chloroperoxidase reactivities. A typical peroxidative reaction and heteroatom dealkylation, common to both O2 activating enzymes, were also studied. Bleomycin was found to catalyze peroxidation of o-dianisidine. The ferric drug complex was found competent in carrying out N-demethylation of N,N-dimethylaniline when peroxides or peroxyacids or iodosobenzene were used as the oxidants. N-Demethylation was not achieved when N,N-dimethylaniline-N-oxide was substituted as the oxidant under similar conditions. Hydroxylation of cumene and decarboxylation of phenylperacetic acid were not found to be catalyzed by bleomycin. Oxygen evolution from m-chloroperbenzoic acid and chlorination of monochlorodimedone from chloride ion and hydrogen peroxide were found to be catalyzed by bleomycin. Cytochrome P-450cam was also evaluated for O2 evolution, and halogenation activity and was found not to demonstrate such reactivities. The results of this initial survey, along with those of previous studies, appear to indicate that the chemical reactivity of bleomycin can be more closely aligned with the reactivities demonstrated by chloroperoxidase than those of cytochrome P-450.     

Genetic regulation of coumarin hydroxylase activity in mice. Biochemical characterization of the enzyme from two inbred strains and their F1 hybrid.

Hydroxylation of coumarin to 7-hydroxycoumarin by liver microsomes from control or phenobarbital-pretreated mice is 5- to 10-fold higher in the DBA/2J strain compared to the AKR/J strain, while activities of nine other cytochrome P-450 mediated oxidations show only minor differences. Mixing experiments with whole liver homogenates and subcellular fractionations do not reveal the presence of enzyme activators or inhibitors or competing enzyme reactions in either strain. Comparisons of pH optima (pH 7.6), heat stability at 52 degrees C (6 to 8 min for 50% inactivation), and Km values (0.45 to 0.50 microM coumarin) for coumarin hydroxylase show no significant differences in the two strains of mice or their F1 hybrid. Similarly, only minor differences in inhibition of coumarin hydroxylase by carbon monoxide, SKF-525A, menadione, and several other inhibitors of microsomal mixed function oxidase reactions are observed in the two strains. In contrast to these data, aniline and metyrapone, two compounds which bind to the heme iron of cytochrome P-450 to form ferrihemochromes, show differential and opposite patterns of inhibition of enzyme activity in the DBA/2J and AKR/J mouse strains. This latter observation suggests that a structurally different cytochrome P-450 may hydroxylate coumarin in these two inbred mouse strains.     

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.     

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

One-electron reduction of the oxyform of 2,4-diacetyldeuterocytochrome P-450cam

The reaction of the hydrated electron with a ferrous oxygenated form of modified cytochrome P-450cam, containing 2,4-diacetyldeuteroheme, was investigated by the use of pulse radiolysis. The ferrous oxygenated form of this enzyme was reduced by hydrated electrons to form the product, which exhibits absorption maximum at 470 and 370 nm. From the spectrum obtained, the oxidation state of the product is discussed in relation to the higher oxidation states of chloroperoxidase.     

Thermodynamic properties of oxidation-reduction reactions of bacterial, microsomal, and mitochondrial cytochromes P-450: an entropy-enthalpy compensation effect

An optically transparent thin-layer electrode cell with a very small volume was used for determination of the formal reduction potentials of bacterial, microsomal, and mitochondrial cytochromes P-450. At an extrapolated zero concentration of dye, the bacterial cytochrome from Pseudomonas putida catalyzing the hydroxylation of camphor and the adrenal mitochondrial cytochrome catalyzing the cholesterol side-chain cleavage reaction had formal reduction potentials of -168 and -285 mV (pH 7.5 and 25 degrees C), respectively. The oxidation-reduction potentials for the rabbit liver microsomal cytochrome P-450 induced by 3-methylcholanthrene and the mitochondrial cytochrome for steroid 11 beta-hydroxylation were found as -360 and -286 mV, respectively. Potential measurements at different temperatures allowed documentation of the standard thermodynamic parameters for cytochrome P-450 reduction for the first time. All cytochromes tested were found to have a relatively large negative entropy change upon reduction. The extent of these changes is comparable to that observed for the ferric-ferrous couple of cytochrome c. An entropy-enthalpy compensation effect was observed among the four cytochromes P-450 examined although the correlation is weaker than that observed with cytochrome c isolated from various sources.     

Flash photolysis studies on the CO complexes of ferrous cytochrome P-450scc and cytochrome P-45011 beta. Effects of steroid binding on the photochemical and ligand binding properties

Upon irradiation by a light flash (100-J), the carbon monoxide complex of cytochrome P-450scc was fully photodissociated in both the presence and absence of cholesterol, while less than 20% of the CO complex was photodissociable with those of deoxycorticosterone-bound and -free forms of cytochrome P-45011 beta. When the quantum yield of the reaction was measured for each photodissociable portion, the values were 0.5 and 1.0 for the substrate-free and -bound forms of cytochrome P-450scc, and 0.03 and 0.8 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. Thus, CO complexes of these enzymes become more photosensitive upon binding with the specific substrates. Steroid binding also affected kinetic constants of reactions between the ferrous enzymes and CO. The rate constants for the CO recombination at 15 degrees C were 2.7 X 10(6) and 2.3 X 10(5) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-450scc, and were 7.0 X 10(5) and 5.4 X 10(3) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. The rate constants for the CO dissociation also decreased upon the steroid bindings. The products of the enzyme reactions, pregnenolone and corticosterone, had similar effects on the kinetic constants. From these findings, we postulate that the binding of a steroid to the substrate site of each enzyme alters the bonding character of CO with the heme-iron, thereby affecting both photochemical and kinetic properties of the CO complex. The nature of the photoindissociable portion of the CO complex of cytochrome P-45011 beta is also discussed.     

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

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

Mechanisms of cytochrome P450 and peroxidase-catalyzed xenobiotic metabolism.

The cytochrome P450 enzyme systems catalyze the metabolism of a wide variety of naturally occurring and foreign compounds by reactions requiring NADPH and O2. Cytochrome P450 also catalyzes peroxide-dependent hydroxylation of substrates in the absence of NADPH and O2. Peroxidases such as chloroperoxidase and horseradish peroxidase catalyze peroxide-dependent reactions similar to those catalyzed by cytochrome P450. The kinetic and chemical mechanisms of the NADPH and O2-supported dealkylation reactions catalyzed by P450 have been investigated and compared with those catalyzed by P450 and peroxidases when the reactions are supported by peroxides. Detailed kinetic studies demonstrated that chloroperoxidase- and horseradish peroxidase-catalyzed N-demethylations proceed by a Ping Pong Bi Bi mechanism whereas P450-catalyzed O-dealkylations proceed by sequential mechanisms. Intramolecular isotope effect studies demonstrated that N-demethylations catalyzed by P450s and peroxidases proceed by different mechanisms. Most hemeproteins investigated catalyzed these reactions via abstraction of an alpha-carbon hydrogen whereas reactions catalyzed by P-450 and chloroperoxidase proceeded via an initial one-electron oxidation followed by alpha-carbon deprotonation. 18O-Labeling studies of the metabolism of NMC also demonstrated differences between the peroxidases and P450s. Because the hemeprotein prosthetic groups of P450, chloroperoxidase, and horseradish peroxidase are identical, the differences in the catalytic mechanisms result from differences in the environments provided by the proteins for the heme active site. It is suggested that the axial heme-iron thiolate moiety in P450 and chloroperoxidase may play a critical role in determining the mechanism of N-demethylation reactions catalyzed by these proteins.     

M?ssbauer investigations of high-spin ferrous heme proteins. II. Chloroperoxidase, horseradish peroxidase, and hemoglobin.

Reduced samples of chloroperoxidase, horseradish peroxidase, and deoxyhemoglobin were studied by M?ssbauer spectroscopy in strong magnetic fields. The intricate paramagnetic spectra of chloroperoxidase were evaluated in detail in the framework of a spin Hamiltonian pertinent to high-spin ferrous iron. The studies strongly suggest that, in their reduced states, chloroperoxidase from Caldariomyces fumago and cytochrome P-450 from Pseudomonas putida have similar, if not identical ligand structures of the heme iron. The spectral similarities of these two proteins, noted in an earlier M?ssbauer investigation, are further explored and substantiated. Reduced horseradish peroxidase and deoxyhemoglobin, on the other hand, show high-field M?ssbauer spectra that differ considerably from each other and, in particular, from those of the P-450 type, suggesting a different ligand arrangement of the heme iron for each case.     

Spin state transitions of liver microsomal cytochrome P-450.

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.     

Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase, and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction, studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different. As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..     

A new microphotometric method for measurement of cytochrome P-450 in sections of liver

We developed a new microphotometric method for measuring the amounts of cytochrome P-450 (P-450) in fresh frozen sections of liver. Four serial frozen sections cut from the liver were separately incubated in 50 mM Tris-HCl buffer (pH 8.0) alone, in buffer containing sodium dithionite, in buffer saturated with carbon monoxide (CO), and in buffer saturated with CO and containing sodium dithionite. The difference between absorbance at 450 nm and that at 490 nm was measured in these sections with a simple microphotometer system. This method yielded precise amounts of P-450 in sections by measuring the true extinction of P-450 and by minimizing the effect of contaminating hemoproteins. Livers of adult rats contained large amounts of P-450, which was greater in perivenular hepatocytes than in periportal hepatocytes. In livers of newborn rats, however, small amounts of the enzyme were distributed evenly throughout the lobule.     

Proton activities for three states of cytochrome P-450cam.

Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the $\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.