Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450

Phenylacetylene and biphenylacetylene are oxidized by cytochrome P-450 to the corresponding arylacetic acids. The acetylenic hydrogen shifts to the adjacent carbon and one atom of molecular oxygen is incorporated into the carboxylic acid group in these transformations, which are subject to a large kinetic isotope effect when the acetylenic hydrogen is replaced by deuterium. The same products and isotope effects are observed when the two arylacetylenes are oxidized by m-chloroperbenzoic acid rather than by the enzyme. In contrast, the inactivation of cytochrome P-450 that occurs during the oxidation of phenylacetylene is insensitive to deuterium substitution. The partition ratio between metabolite formation and enzyme inactivation consequently changes from 26 to 15 in going from phenylacetylene to the deuterated analogue. Metabolite formation therefore diverges from heme alkylation very early in the catalytic process.     

Cytochrome P-450-catalyzed hydroxylation and carboxylic acid ester cleavage of Hantzsch pyridine esters

Cytochrome P-450 (P-450)-catalyzed oxidation of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester gives rise to 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester and to 2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester, identified in this work. A pyridine hydroxymethyl diester of the sort of the latter compound is novel; under acidic or dehydrating conditions the diester is readily converted to a cyclic lactone (2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid 5-ethyl ester lactone). 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester was not hydroxylated to form this hydroxymethyl compound or lactone, but 1,4-dihydro-2-hydroxymethyl-4-phenyl-6-methyl-3,5-pyridinedicarboxyli c acid diethyl ester was enzymatically oxidized to give both products. The rates of oxidative carboxylic ester cleavage and methyl hydroxylation varied among individual forms of P-450 tested. Experiments with 2H and 3H labels were used to estimate an intrinsic kinetic deuterium isotope effect of 15 for ethyl ester cleavage by rat liver P-450PB-B in a reconstituted system. Rat liver microsomal systems showed kinetic deuterium and tritium isotope effects of 8 and 11, respectively, and this deuterium isotope effect was not attenuated in either intra- or intermolecular competitive experiments. When deuterium was present in the ethyl (ester) groups, increases in the rate of 2-methyl hydroxylation were observed in rat liver microsomes and with purified P-450 beta NF-B (but not with P-450PB-B). Deuteration of the methyl groups gave rise to kinetic isotope effects of 7-11, but no increases were seen in the rates of ester cleavage. These studies and those on rates of substrate disappearance indicate that isotopically sensitive branching (metabolic switching) observed in these systems is not necessarily bidirectional.     

Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin

The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.     

Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects

The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.     

Kinetic mechanism and nucleotide specificity of NADH peroxidase

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.     

Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.     

Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3-hydroxynorcamphor product.

Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1-methyl-norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1-methyl-norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1-methyl-norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.     

Theoretical study of the product specificity in the hydroxylation of camphor, norcamphor, 5,5-difluorocamphor, and pericyclocamphanone by cytochrome P-450cam

The hydroxylations of d-camphor, norcamphor, pericyclocamphanone, and 5,5-difluorocamphor by cytochrome P-450cam have been examined using theoretical methods to identify and characterize properties which determine product specificity. Experimental results indicate that each molecule is hydroxylated with quite different regio-specificity when metabolized by P-450cam. This result is surprising in view of their overall structural similiarity. Herein we report the results of calculations on d-camphor and three of its analogues which suggest that all of these molecules should, when metabolized by P-450cam, form hydroxylation products and predict the product distribution for each. Our conclusions are based on two fundamental criteria which are consistent with a generally accepted radical mechanism in determining product specificity in these molecules: 1) relative heats of formation of the radicals formed by abstracting a hydrogen, and 2) orientation of the substrate molecule with respect to the putative active oxygen species bound to iron. Our results explain the experimental observations for camphor and 5,5-difluorocamphor but disagree with original published results for norcamphor and pericyclocamphanone. In light of our results, new experiments have been performed for norcamphor and the original data reexamined for pericyclocamphanone. Our predictions have recently been experimentally confirmed for norcamphor, and unpublished data (Dr. S. Sligar) suggest that the same is true for pericyclocamphanone.     

Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.     

Oxidative cleavage of carboxylic esters by cytochrome P-450

Cytochrome P-450 was demonstrated to catalyze the oxidative cleavage of carboxylic acid esters to the corresponding carboxylic acids. 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester and related dialkyl esters were shown to serve as substrates in NADPH-fortified rat liver microsomes and reconstituted systems containing purified cytochrome P-450 enzymes. The ethyl group gave rise to acetaldehyde. The reactions proceed with large kinetic deuterium isotope effects, consistent with the view that P-450 abstracts a hydrogen atom in the mechanism. Oxygen rebound to the radical site is then postulated to complete the reaction and lead to a hemiacetal-like structure which collapses to give the products. Rate studies with differing alkyl substituents showed that the reaction was more rapid with removal of an ethyl than a methyl or isopropyl group, consistent with the view that the ethyl optimizes steric and inductive effects. Oxidative cleavage of carboxylic acid esters has little biochemical precedent, due to the difficult character of the reaction, and should be considered as an alternative to direct hydrolysis.     

Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.     

Isotope effects and intermediates in the reduction of NO by P450(NOR)

The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450(NOR)) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,4(2)H(2)]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the Fe(III)-NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7+/-0.4 for the R-form and 1.1+/-0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric-NO complex at -10 degrees C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450(NOR). Based on these results we postulate hydride transfer from NADH to the ferric P450-NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe-hydroxylamine complex or by electron transfer from the NO radical to the ferric-thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions.     

Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.     

Mechanistic studies with purified components of the liver microsomal hydroxylation system: spectral intermediates in reaction of cytochrome P-450 with peroxy compounds.

Recent investigations in this laboratory on the mechanism of action of liver microsomal cytochrome P-450 (P-450 LM) and its interaction with other components of the hydroxylation system are presented. Two electrophoretically homogeneous forms of the cytochrome, phenobarbital-inducible P-450 LM2 and 5,6-benzoflavone-inducible P-450 LM4, so designated according to their relative electrophoretic mobilities, were used in these studies. Phosphatidylcholine is required in the reconstituted enzyme system for rapid electron transfer from NADPH to P-450 LM, catalyzed by NADPH-cytochrome P-450 reductase, as well as for maximal hydroxylation activity with either molecular oxygen or a peroxy compound serving as oxygen donor to the substrate. The phospholipid facilitates the binding of both substrate and reductase to P-450 LM and apparently causes a structural change in the cytochrome as shown by an increase in alpha-helical content, determined by circular dichroic spectrometry. P-450LM3 and LM4 are one-electron acceptors under anaerobic conditions, in accord with previous potentiometric titrations and product yield data, but in disagreement with previous titrations with reducing agents. The cause for the discrepancy between the present and earlier results is not yet fully understood. Stopped flow spectrophotometry was employed to detect intermediates in the reaction of peroxy compounds with P-450LM2. With m-chloroperbenzoic acid the intermediate formed has absorption maxima at 375, 425, and 540 nm in the absolute spectrum and at 370, 436, and 540 nm in the difference spectrum (intermediate minus oxidized form). A study of the magnitude of the spectral change at various peracid concentrations indicated that with this oxidant the reaction shows a dependence resembling a binding curve. These and other experiments with various oxidants, including cumente hydroperoxide, suggest a reversible two-step mechanism according to the reaction: P-450 LM + oxidant equilibrium C equilibrium D, where C may be an enzyme-oxidant complex and D is a spectral intermediate of unknown structure. A scheme is proposed for the mechanism of action of P-450 LM based on these and earlier studies, including evidence from deuterium isotope experiments for the formation of a substrate carbon radical prior to oxygen transfer.     

A radiometric kynurenine monooxygenase assay

Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes L-[3H]kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro-R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.     

The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450CAM

The crystal structures of cytochrome P-450CAM complexed with the alternative substrates norcamphor and adamantanone have been refined at 2.0-A resolution and compared with the native, camphor-bound form of the enzyme. Norcamphor lacks the 8-, 9-, and 10-methyl groups of camphor. Thus, specific interactions between these groups and phenylalanine 87 and valines 247 and 295 are missing in the norcamphor complex. As a result, norcamphor binds about 0.9 A further from the oxygen-binding site than does camphor, which allows sufficient room for a water molecule or hydroxide ion to remain coordinated with the heme iron atom. The larger adamantanone occupies a position closer to that of camphor and, as in the camphor-bound enzyme, the heme iron remains pentacoordinate with no solvent molecule coordinated as a sixth ligand. A comparison of crystallographic temperature factors indicates that norcamphor is more "loosely" bound than are either camphor or adamantanone, as might be expected from the relative sizes of the different substrates. The looser fit of norcamphor in the active-site pocket results in a less specific pattern of hydroxylation. The presence of an aqua ligand is the likely structural basis for the norcamphor-P-450CAM complex having both a lower redox potential and higher percentage of low-spin heme than do either the camphor-P-450CAM or adamantanone-P-450CAM complexes.     

Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines

A variety of different 4-substituted 1,4-dihydropyridine Hantzsch esters are substrates for ring dehydrogenation by a cytochrome P-450 (P-450) enzyme (P-450 UT-A); the substitutent could be varied from a hydrogen to a naphthalenyl, but a pyrenyl derivative was not dehydrogenated. When a 4-alkyl group is present, both the P-450 which oxidizes the substrate and other P-450s can be inactivated (by putative alkyl radicals). P-450s did not discriminate with regard to removal of the 4-H atoms from an enantiomeric pair of dihydropyridines. Losses of the 4-proton and N-methyl from a N-methyl-1,4-dihydropyridine occur at similar rates. The calculated intrinsic kinetic hydrogen isotope effect (Dk) for dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid dimethyl ester was 2.9 in a reconstituted P-450 UT-A enzyme system. No significant kinetic hydrogen isotope effect was observed in microsomal incubations for the dehydrogenation of this compound or 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester in a variety of competitive and noncompetitive experiments. In light of previous studies on the magnitude of kinetic hydrogen isotope effects in P-450 systems (e.g. Miwa et al., 1983 (Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449], the mechanistic proposals of Augusto et al., 1982 (Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Biol. Chem. 257, 11288-11295)) for enzyme inactivation by 4-alkyl-substituted Hantzsch pyridine esters, and other precedents for sequential electron transfer in amine oxidation by P-450s, we interpret these results as being consistent with P-450-mediated 1-electron oxidation of dihydropyridines followed by the facile loss of the 4-proton, with subsequent electron transfer to complete the reaction.     

Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin.

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.