On the regiospecificity of vanadium bromoperoxidase.

Vanadium haloperoxidase enzymes catalyze the oxidation of halide ions by hydrogen peroxide, producing an oxidized intermediate, which can halogenate an organic substrate or react with a second equivalent of hydrogen peroxide to produce dioxygen. Haloperoxidases are thought to be involved in the biogenesis of halogenated natural products isolated from marine organisms, including indoles and terpenes, of which many are selectively oxidized or halogenated. Little has been shown concerning the ability of the marine haloperoxidases to catalyze regioselective reactions. Here we report the regiospecific bromoperoxidative oxidation of 1,3-di-tert-butylindole by V-BrPO from the marine algae Ascophyllum nodosum and Corallina officinalis. Both enzymes catalyze the regiospecific oxidation of 1,3-di-tert-butylindole in a reaction requiring both H(2)O(2) and Br(-) as substrates, but which produce the unbrominated 1,3-di-tert-butyl-2-indolinone product exclusively, in near quantitative yield (i.e. one H(2)O(2) consumed per product). By contrast, reactions with the controlled addition of aqueous bromine solution (HOBr = Br(2) = Br(3)(-)) produce three monobromo and one dibromo-2-indolinone products, all of which differ from the V-BrPO-catalyzed product. Further, reactivities of 1,3-di-tert-butyl-2-indolinone with both aqueous bromine and V-BrPO differ significantly and shed light onto the possible nature of the oxidizing intermediate. This is the first example of a regiospecific bromination by a vanadium haloperoxidase and further extends their usefulness as catalysts.

Reversible pressure deformation of a thermophilic cytochrome P450 enzyme (CYP119) and its active-site mutants.

The pressure stability of the thermophilic CYP119 from Sulfolobus solfataricus and its active-site Thr213 and Thr214 mutants was investigated. At 20 degrees C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol. The volume of activation of the process was -9.5 mL/mol. The inactivation transition was retarded, and the absolute reaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity. High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480 MPa. The effect was reversible and suggested considerable contraction of the protein. Aerobic decomposition of iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variation of the N-arylprotoporphyrin-IX regioisomer (N(B):N(A):N(C):N(D)) adduct pattern from 39:47:07:07 at 0.1 MPa to 23:36:14:27 at 400 MPa. Preincubation of the protein at 400 MPa followed by complex formation and decomposition gave the same regioisomer distribution as untreated protein. The results indicate that the protein is reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450(cam) and other P450 enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions.

An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates.

Cytochrome P450(eryF) (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450(eryF) by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha-, 12-, and 16alpha-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450(eryF)-androstenedione complex. P450(eryF) is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450(eryF) mutants are also attractive templates for the engineering of novel P450 activities.

The active site of the thermophilic CYP119 from Sulfolobus solfataricus.

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.

Protein control of the formation and decomposition of the CYP119 and CYP101 aryl-iron complexes.

CYP119, the first thermophilic P450 enzyme, reacts much more slowly than CYP101 (P450cam) with aryldiazenes to give sigma-bonded aryl-iron complexes. The CYP119 complexes are stable anaerobically at 80 degrees C but are readily oxidized by O2 to give the N-arylprotoporphyrin IX regioisomers. The aryl shift can also be initiated in the absence of O2 by K3Fe(CN)6. In contrast, the corresponding CYP101 complexes are insensitive to O2 but decompose at temperatures above 50 degrees C owing to denaturation of the protein. The rate of the CYP119 aryl shift is decreased by electron-withdrawing substituents, with rho = -1.50 for both the O2- and K3Fe(CN)6-dependent reactions. A similar dependence (rho = -0.90) is observed for the K3Fe(CN)6-dependent CYP101 shift. The enthalpies and entropies of activation suggest that the CYP119 and CYP101 K3Fe(CN)6-mediated reactions are similar, but the CYP119 O2-dependent reaction proceeds via a different transition state. In all cases, the rate-determining step is oxidation of the aryl-iron complex. The temperature dependence of the O2- and K3Fe(CN)6-dependent CYP119 shifts provides evidence for temperature-dependent equilibration of two active site conformations. The oxygen sensitivity of the CYP119 aryl-iron complexes, and the temperature dependence of their rearrangement, reflect the unique active site properties of this thermophilic P450 enzyme.

Site-specific spin trapping of tyrosine radicals in the oxidation of metmyoglobin by hydrogen peroxide.

The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

Peroxidation of a specific tryptophan of metmyoglobin by hydrogen peroxide.

Globin-centered radicals at tyrosine and tryptophan residues and a peroxyl radical at an unknown location have been reported previously as products of the reaction of metmyoglobin with hydrogen peroxide. The peroxyl radical is shown here to be localized on tryptophan through the use of recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3. Peroxyl radical formation was not prevented by site-directed mutations that replaced all three tyrosines, the distal histidine, or tryptophan 7 with non-oxidizable residues. In contrast, mutation of tryptophan 14 prevents peroxyl radical formation, implicating tryptophan 14 as the specific site of the peroxidation.

Protein radicals in myoglobin dimerization and myoglobin-catalyzed styrene epoxidation.

Two mechanisms have been identified for the H2O2-dependent epoxidation of styrenes by sperm whale myoglobin (Mb) [S. Rao, A. Wilks, and P. R. Ortiz de Montellano, J. Biol. Chem. 268, 803-908 (1993)]: (a) ferryl (FeIV = O) oxygen transfer with retention of stereochemistry and incorporation of an oxygen from H2O2, and (b) protein peroxy radical cooxidation with loss of stereochemistry and incorporation of an oxygen from O2. As shown here, cis-beta-methylstyrene is preferentially oxidized to the trans-epoxide when the H2O2:Mb ratio is <0.5 but increasingly to the cis-isomer as the ratio increases to and above 1. At a high (4:1) H2O2:Mb ratio, both the absolute yield and the cis:trans-epoxide ratio increase in proportion to the cis-beta-methylstyrene concentration. A protein radical formed in the Mb-H2O2 reaction also causes dimer and trimer formation, maximum dimer formation (approximately 30%) being obtained with 1 equivalent of H2O2. At low H2O2:Mb ratios, the oxidation equivalents utilized for protein oligomerization and styrene oxidation account for the available H2O2. Previous studies have shown that His-64 is important for protein-mediated olefin cooxidation and Tyr-151/Tyr-103 for Mb dimerization. The W7F, W14F, and W7F/W14F Mb mutants have now been prepared and the W14F, but not W7F, mutation shown to modestly decrease cooxidation of cis-beta-methylstyrene to the trans-epoxide. Neither tryptophan mutation alters dimer formation. Dimer formation is modestly increased rather than decreased by styrene, suggesting that styrene cooxidation and dimerization do not compete. The results indicate that (a) cis-beta-methylstyrene cooxidation and protein dimerization, both of which are mediated by protein radicals, are favored at low H2O2:Mb ratios, (b) as the H2O2:Mb ratio increases, the ferryl epoxidation pathway surpasses the cooxidation mechanism, (c) Trp-14 but not Trp-7 influences olefin cooxidation, and (d) different, possibly nonequilibrating, radicals mediate olefin cooxidation and protein dimerization.

Active-site topologies of human CYP2D6 and its aspartate-301 --> glutamate, asparagine, and glycine mutants.

Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5-7 angstroms from the site of the oxidation. The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, and p-biphenyldiazene, which react with P450 enzymes to form sigma-bonded aryl-iron (Fe-Ar) complexes. Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces the N-arylprotoporphyrin IX regioisomers (NB:NA:NC:ND, in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; and p-biphenyl, 16:84:00:00. These results suggest that the CYP2D6 active site is open above pyrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D. This geometry differs from those determined by the same method for P450s for which crystal structures are available. Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in the N-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity. Replacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually abolishes catalytic activity. These results provide a refined model of the active site of CYP2D6. They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ring B, but not to major structural reorganization of the active site.

Chloroperoxidase-catalyzed benzylic hydroxylation.

Chloroperoxidase oxidizes p-methylanisole and p-ethylanisole to 4-methoxybenzyl alcohol and 1-(4'-methoxyphenyl)ethanol, respectively. It ineffectively oxidizes toluene to benzyl alcohol but does not appear to oxidize toluene substituted with strong electron-withdrawing groups. O-Demethylation is also observed. The enzyme is sensitive to substituents at other than the para position and does not detectably catalyze benzylic hydroxylation of p-methylanisole if it bears additional methyl or methoxy groups. An exception is 1,2-(methylenedioxy)-4-methylbenzene, which is oxidized to both 3,4-(methylenedioxy)benzyl alcohol and 2-hydroxy-4-methylphenol. Studies with H2(18)O2 indicate that all the oxygen incorporated into the product in the oxidation of p-methylanisole to 4-methoxybenzyl alcohol derives from the peroxide. The mono- and dideuterated methyl analogues of p-methylanisole are oxidized with apparent intramolecular isotope effects of 3.51 and 3.34, respectively. Abstraction of a hydrogen from a carbon bearing a hydroxyl group competes effectively with benzylic oxidation because 2-[1,1-2H2]phenylethanol is oxidized to 2-[1-2H]- rather than 2-[1,2-2H2]phenylacetaldehyde. Aldehyde formation therefore involves abstraction of the carbinol hydrogen rather than hydrogen migration to a benzylic carbocation intermediate. Chloroperoxidase resembles cytochrome P450 in that it catalyzes benzylic hydroxylation reactions but it has a more limited substrate specificity.