Probing the Structure-Function Relationship of a Multifunctional Enzyme using Crystallographic Diffraction Methods (original) (raw)
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X-ray crystal structures of dehaloperoxidasehemoglobin A (DHP A) from Amphitrite ornata soaked with substrate, 2,4,6-tribromophenol (2,4,6-TBP), in buffer solvent with added methanol (MeOH), 2-propanol (2-PrOH), and dimethyl sulfoxide (DMSO) reveal an internal substrate binding site deep in the distal pocket above the α-edge of the heme that is distinct from the previously determined internal inhibitor binding site. The peroxidase function of DHP A has most often been studied using 2,4,6-trichlorophenol (2,4,6-TCP) as a substrate analogue because of the low solubility of 2,4,6-TBP in an aqueous buffer solution. Previous studies at low substrate concentrations pointed to the binding of substrate 2,4,6-TCP at an external site near the exterior heme βor δ-edge as observed in the class of heme peroxidases. Here we report that the turnover frequencies of both substrates 2,4,6-TCP and 2,4,6-TBP deviate from Michaelis−Menten kinetics at high concentrations. The turnover frequency reaches a maximum in the range of 1400−1700 μM, with a decrease in rate at higher concentrations that is both substrate-and solvent-dependent. The X-ray crystal structure is consistent with the presence of an internal active site above the heme α-edge, in which the substrate would be oxidized in two consecutive steps inside the enzyme, followed by attack by H 2 O via a water channel in the protein. The physiological role of the internal site may involve interactions with any of a number of aromatic toxins found in benthic ecosystems where A. ornata resides.
Journal of Biological Chemistry, 2000
The full-length, protein coding sequence for dehaloperoxidase was obtained using a reverse genetic approach and a cDNA library from marine worm Amphitrite ornata. The crystal structure of the dehaloperoxidase (DHP) was determined by the multiple isomorphous replacement method and was refined at 1.8-Å resolution. The enzyme fold is that of the globin family and, together with the amino acid sequence information, indicates that the enzyme evolved from an ancient oxygen carrier. The peroxidase activity of DHP arose mainly through changes in the positions of the proximal and distal histidines relative to those seen in globins. The structure of a complex of DHP with 4-iodophenol is also reported, and it shows that in contrast to larger heme peroxidases DHP binds organic substrates in the distal cavity. The binding is facilitated by the histidine swinging in and out of the cavity. The modeled position of the oxygen atom bound to the heme suggests that the enzymatic reaction proceeds via direct attack of the oxygen atom on the carbon atom bound to the halogen atom.
The time-resolved kinetics of substrate oxidation and cosubstrate H 2 O 2 reduction by dehaloperoxidase-hemoglobin (DHP) on a seconds-to-minutes time scale was analyzed for peroxidase substrates 2,4,6-tribromophenol (2,4,6-TBP), 2,4,6-trichlorophenol (2,4,6-TCP), and ABTS. Substrates 2,4,6-TBP and 2,4,6-TCP show substrate inhibition at high concentration due to the internal binding at the distal pocket of DHP, whereas ABTS does not show substrate inhibition at any concentration. The data are consistent with an external binding site for the substrates with an internal substrate inhibitor binding site for 2,4,6-TBP and 2,4,6-TCP. We have also compared the kinetic behavior of horseradish peroxidase (HRP) in terms of k cat , K m AH 2 and K m H 2 O 2 using the same kinetic scheme. Unlike DHP, HRP does not exhibit any measurable substrate inhibition, consistent with substrate binding at the edge of heme near the protein surface at all substrate concentrations. The binding of substrates and their interactions with the heme iron were further compared between DHP and HRP using a competitive fluoride binding experiment, which provides a method for quantitative measurement of internal association constants associated with substrate inhibition. These experiments show the regulatory role of an internal substrate binding site in DHP from both a kinetic and competitive ligand binding perspective. The interaction of DHP with substrates as a result of internal binding actually stabilizes that protein and permits DHP to function under conditions that denature HRP. As a consequence, DHP is a tortoise, a slow but steady enzyme that wins the evolutionary race against the HRP-type of peroxidase, which is a hare, initially rapid, but flawed for this application because of the protein denaturation under the conditions of the experiment.
Biochemistry, 2018
The dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete Amphitrite ornata is a multifunctional hemoprotein that catalyzes the oxidation of a wide variety of substrates, including halo-/nitro-phenols, haloindoles, and pyrroles, via peroxidase and/or peroxygenase mechanisms. To probe whether substrate substituent effects can modulate enzyme activity in DHP, we investigated its reactivity against a panel of o-guaiacol substrates given their presence (from native/halogenated and non-native/anthropogenic sources) in the benthic environment that A. ornata inhabits. Using biochemical assays supported by spectroscopic, spectrometric, and structural studies, DHP was found to catalyze the H2O2-dependent oxidative dehalogenation of 4-haloguaiacols (F, Cl, Br) to 2-methoxybenzoquinone (2-MeOBQ). 18O-labeling studies confirmed that O-atom incorporation was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The 2-MeOBQ product furthe...
Biochemistry, 2006
Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H 2 O 2 . This observation suggests that the mechanism of DHP relies on H 2 O 2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products. FIGURE 1: Superposition of the DHP and Mb structures using the heme ring atoms as the common atoms. A high-resolution SWMb X-ray structure (69) and the DHP structure 1EW6 (4) were retrieved from the Protein Data Bank. The superposition was carried out using Insight II (Accelrys, Inc.). (A) Similarity of the helical structure of DHP and SWMb with an offset of 1.5 Å relative to the heme ring atoms. (B) Similarity of the residues in the distal pocket. In the closed conformation, both the valine and histidine are present in the same relative orientation; however, the corresponding residues SWMb-H64/DHP-H55 and SWMb-V68/DHP-V59 are shifted by 1.5 Å relative to the heme iron. The open conformation is also recorded in the DHP X-ray crystal structure (1EW6), which has two sets of coordinates for H55.
Peroxygenase and Oxidase Activities of Dehaloperoxidase-Hemoglobin from Amphitrite ornata
The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H 2 O 2dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H 2 O 2 , indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globinactive oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H 2 O 2 also yielded 5,5′-Br 2 -indigo above the expected reaction stoichiometry under aerobic conditions, and O 2 -concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5′-Br 2 -indigo, and together suggest a newly identified oxidase activity for DHP.
Distal histidine conformational flexibility in dehaloperoxidase fromAmphitrite ornata
Acta Crystallographica Section D Biological Crystallography, 2008
ZUXU, CHEN. Distal Histidine Conformational Flexibility in Dehaloperoxidase from Amphitrite Ornata. (Under the direction of Stefan Franzen.) The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein, which has a globin fold, but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting para-halogenated phenols to the corresponding quinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Herein, we report that the distal histidine shows a large conformational flexibility in the deoxy form. Crystals of deoxy ferrous DHP were obtained by reducing the ferric wild-type DHP in sodium dithionite solution and the structure was determined at 100K to a resolution of 1.22Å. The heme iron in the deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.23 Å for the heme iron relative to the oxy form. The distal histidine, H55 is observed in conformations, which are analogous to the open and closed forms of myoglobin. In the closed conformation, H55 is located inside the distal pocket, but does not penetrate as deeply into the distal pocket as in the metaquo ferric or oxy ferrous structures. This observation is consistent with the hypothesis that H55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. There are two open or solvent-exposed conformations, in which H55 is more than 9.5 Å away from the heme. The comparison of the deoxy structure with the other structures provides new insight into the correlation between the heme iron ligation and the conformation of distal histidine in the DHP.
Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases
Science (New York, N.Y.), 2016
The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different.