Gregory M. Raner

The Mechanism of Oxidative Halophenol Dehalogenation by Amphitrite ornata Dehaloperoxidase Is Initiated by H2O2 Binding and Involves Two Consecutive One-Electron Steps: Role of Ferryl Intermediates

The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.

Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes

The reaction of hydrogen peroxide and certain aromatic aldehydes with cytochrome P450BM3-F87G results in the covalent modification of the heme cofactor of this monooxygenase. Analysis of the resulting heme by electronic absorption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P450(2B4), which apparently occurs via a peroxyhemiacetal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was observed that replacement of the Phe-87 in the P450BM3 by the smaller glycyl residue was essential for the modification to proceed, as the wild-type enzyme showed no spectral changes under identical conditions. The kinetics of this reaction were examined by stopped-flow spectrophotometry with 3-phenylpropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the process of heme modification was biphasic, with initial bleaching of the Soret absorbance, followed by an increase in absorbance centered at 430 nm, consistent with meso-heme adduct formation. The intermediate formed during phase I also showed an increased absorbance between 700 and 900 nm, relative to the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for phase II. All of these observations are consistent with a mechanism involving radical attack at the gamma-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of which produces the gamma-meso-alkyl heme derivative.

Separation of protein inclusion bodies from Escherichia coli lysates using sedimentation field‐flow fractionation

Sedimentation field-flow fractionation has been used to separate my- ohemerythrin inclusion bodies from components of growth media, soluble proteins, and unlysed cells that are present in Escherichia coli cell lysates. Collected . fractions were concentrated and then analyzed by sodium dodecyl sulfate SDS polyacrylamide gel electrophoresis to confirm the presence of myohemerythrin inclusion bodies and to determine their position in the elution sequence. The fractograms of samples prepared using two different cell lysing methods were compared. Q 1997 John Wiley & Sons, Inc. JM icro Sep9: 233)239, 1997

Functional Role of Leucine-103 in Myohemerythrin†

Hemerythrins (Hrs) and myohemerythrins (Mhrs) are nonheme iron proteins that function as O2 carriers in four marine invertebrate phyla. Available amino acid sequences and X-ray structures indicate that a conserved leucine, residue 103 in the Themiste zostericola Mhr sequence, occupies a site distal to the Fe-O-Fe center. The side-chain methyl groups of the analogous leucine in Themiste dyscrita oxyHr are in van der Waals contact with bound O2 in the X-ray crystal structure, and this residue may therefore play a role in stabilizing bound dioxygen with respect to autoxidation. In order to test this hypothesis, the gene for T. zostericola Mhr was synthesized and expressed in Escherichia coli. Two mutant Mhrs, L103V and L103N, were also prepared. Optical spectra and kinetics data for these three proteins are presented. Importantly, neither mutant forms a stable oxy adduct; instead, rapid autoxidation results in formation of the corresponding met forms. In addition, the L103N Mhr displays unusually rapid reduction kinetics, suggesting that the amide functionality of Asn-103 destabilizes most bound ligands and additionally promotes rapid semi-metR <==> semi-metO isomerization.

Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: Toward a functional halophenol bioremediation catalyst

Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam. Toxicol. 34 (1998) 217-222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions (i.e., without excess H(2)O(2)) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observation is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a halophenol bioremediation catalyst.

Isotopic labeling of the heme cofactor in cytochrome p450 and other heme proteins.

A recombinant bacterial expression system that generates 13C-labeled heme or 15N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which the HemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with 13C or 15N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, along with one peroxidase. Confirmed both 13C- and 15N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.

Inhibition of human cytochrome P450 2E1 and 2A6 by aldehydes: Structure and activity relationships

The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ± 0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ± 1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5-12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8 ± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0 ± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1.

Effects of Green Tea Extract on Gene Expression in Human Hepatoma (HepG2) and Tongue Carcinoma (Cal-27) Cells

Some of the beneficial health effects of green tea extract can be traced to the ability of certain constituents in the extract to regulate gene expression. For example, green tea and the major green tea catechins have been shown to induce a variety of gene products related to anti-oxidant or other protective functions. The full range of genes that are activated by this pathway is still being explored, however, the molecular mechanisms involved have been reasonably well characterized. In addition to anti-oxidant genes, a number of genes involved in apoptosis have also been shown to respond to GTE, through both induction and repression. It is likely that the anti-proliferative/anti-cancer effects of GTE may be expressed, in part, via this mechanism. Finally, genes involved in drug metabolism, in particular, cytochrome P450 enzymes, are regulated by GTE, which may have implications for development of chemoprentive strategies using GTE.