@article{schkolnik_utesch_zhao_jiang_thompson_mroginski_hildebrandt_franzen_2013, title={Catalytic efficiency of dehaloperoxidase A is controlled by electrostatics - application of the vibrational Stark effect to understand enzyme kinetics}, volume={430}, ISSN={["1090-2104"]}, DOI={10.1016/j.bbrc.2012.12.047}, abstractNote={The vibrational Stark effect is gaining popularity as a method for probing electric fields in proteins. In this work, we employ it to explain the effect of single charge mutations in dehaloperoxidase-hemoglobin A (DHP A) on the kinetics of the enzyme. In a previous communication published in this journal (BBRC 2012, 420, 733-737) it has been shown that an increase in the overall negative charge of DHP A through mutation causes a decrease in its catalytic efficiency. Here, by labeling the protein with 4-mercaptobenzonitrile (MBN), a Stark probe molecule, we provide further evidence that the diffusion control of the catalytic process arises from the electrostatic repulsion between the enzyme and the negatively charged substrate. The linear correlation observed between the nitrile stretching frequency of the protein-bound MBN and the catalytic efficiency of the single-site mutants of the enzyme indicates that electrostatic interactions play a dominant role in determining the catalytic efficiency of DHP A.}, number={3}, journal={BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, author={Schkolnik, Gal and Utesch, Tillmann and Zhao, Junjie and Jiang, Shu and Thompson, Matthew K. and Mroginski, Maria-Andrea and Hildebrandt, Peter and Franzen, Stefan}, year={2013}, month={Jan}, pages={1011–1015} }
 @article{zhao_de serrano_dumarieh_thompson_ghiladi_franzen_2012, title={The Role of the Distal Histidine in H2O2 Activation and Heme Protection in both Peroxidase and Globin Functions}, volume={116}, ISSN={1520-6106 1520-5207}, url={http://dx.doi.org/10.1021/jp300014b}, DOI={10.1021/jp300014b}, abstractNote={The distal histidine mutations of dehaloperoxidase-hemoglobin A (DHP A) to aspartate (H55D) and asparagine (H55N) have been prepared to study the role played by the distal histidine in both activation and protection against oxidation by radicals in heme proteins. The H55D and H55N mutants of DHP A have ~6-fold and ~11-fold lower peroxidase activities than wild type enzyme toward the oxidation of 2,4,6-trichlorophenol (TCP) to yield 2,6-dichloroquinone (DCQ) in the presence of H(2)O(2). The origin of the lower rate constants may be the solvent-exposed conformations of distal D55 and N55, which would have the dual effect of destabilizing the binding of H(2)O(2) to the heme iron, and of removing the acid-base catalyst necessary for the heterolytic O-O bond cleavage of heme-bound H(2)O(2) (i.e., compound 0). The partial peroxidase activity of H55D can be explained if one considers that there are two conformations of the distal aspartate (open and closed) by analogy with the distal histidine. We hypothesize that the distal aspartate has an active conformation in the distal pocket (closed). Although the open form is observed in the low-temperature X-ray crystal structure of ferric H55D, the closed form is observed in the FTIR spectrum of the carbonmonoxy form of the H55D mutant. Consistent with this model, the H55D mutant also shows inhibition of TCP oxidation by 4-bromophenol (4-BP). Consistent with the protection hypothesis, compound ES, the tyrosyl radical-containing ferryl intermediate observed in WT DHP A, was not observed in H55D.}, number={40}, journal={The Journal of Physical Chemistry B}, publisher={American Chemical Society (ACS)}, author={Zhao, Junjie and de Serrano, Vesna and Dumarieh, Rania and Thompson, Matt and Ghiladi, Reza A. and Franzen, Stefan}, year={2012}, month={Sep}, pages={12065–12077} }
 @article{franzen_thompson_ghiladi_2012, title={The dehaloperoxidase paradox}, volume={1824}, ISSN={1570-9639}, url={http://dx.doi.org/10.1016/j.bbapap.2011.12.008}, DOI={10.1016/j.bbapap.2011.12.008}, abstractNote={The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.}, number={4}, journal={Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics}, publisher={Elsevier BV}, author={Franzen, Stefan and Thompson, Matthew K. and Ghiladi, Reza A.}, year={2012}, month={Apr}, pages={578–588} }
 @article{nicoletti_thompson_franzen_smulevich_2011, title={Degradation of sulfide by dehaloperoxidase-hemoglobin from Amphitrite ornata}, volume={16}, ISSN={["0949-8257"]}, DOI={10.1007/s00775-011-0762-2}, abstractNote={Dehaloperoxidase-hemoglobin (DHP) is a unique multifunctional enzyme with a globin fold. The enzyme serves as the respiratory hemoglobin for the marine worm Amphitrite ornata and has been shown to catalyze the conversion of highly toxic trihalophenols to dihaloquinones as a detoxification function for the organism. Given the simplicity of the structure of A. ornata, it is entirely possible that DHP may play an even more general role in detoxification of the organism from sulfide commonly found in the coastal estuaries where A. ornata thrives. Comparison of DHP with other sulfide-binding hemoglobins shows that DHP possesses several distal cavity structural properties, such as an aromatic cage and a hydrogen-bond-donor amino acid (His55), that facilitate sulfide binding. Furthermore, a complete reduction of the ferric heme occurs after sulfide exposure under aerobic or anaerobic conditions to yield either the oxy or the deoxy ferrous states of DHP, respectively. Oxidation of sulfide by the heme leads to sulfur products that are less toxic to A. ornata. This proposed new function for DHP relies on the highly flexible distal His55 for deprotonation of the bound hydrogen sulfide, similar to H(2)O(2) activation of the peroxidase function, and provides further support for the importance of the flexibility of the distal His55 in this novel globin.}, number={4}, journal={JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, author={Nicoletti, Francesco P. and Thompson, Matthew K. and Franzen, Stefan and Smulevich, Giulietta}, year={2011}, month={Apr}, pages={611–619} }
 @article{thompson_franzen_davis_oliver_krueger_2011, title={Dehaloperoxidase-Hemoglobin from Amphitrite ornata Is Primarily a Monomer in Solution}, volume={115}, ISSN={["1520-6106"]}, DOI={10.1021/jp201156r}, abstractNote={The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 Å(2), an area significantly smaller than the typical value of 1200-2000 Å(2) and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein, which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (~10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K(d) for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.}, number={14}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Thompson, Matthew K. and Franzen, Stefan and Davis, Michael F. and Oliver, Ryan C. and Krueger, Joanna K.}, year={2011}, month={Apr}, pages={4266–4272} }
 @article{d’antonio_d’antonio_de serrano_gracz_thompson_ghiladi_bowden_franzen_2011, title={Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin}, volume={50}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi201368u}, DOI={10.1021/bi201368u}, abstractNote={The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.}, number={44}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={D’Antonio, Edward L. and D’Antonio, Jennifer and de Serrano, Vesna and Gracz, Hanna and Thompson, Matthew K. and Ghiladi, Reza A. and Bowden, Edmond F. and Franzen, Stefan}, year={2011}, month={Nov}, pages={9664–9680} }
 @article{szatkowski_thompson_kaminski_franzen_dybala-defratyka_2011, title={Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: Horseradish peroxidase and dehaloperoxidase}, volume={505}, ISSN={["1096-0384"]}, DOI={10.1016/j.abb.2010.09.018}, abstractNote={The mechanism of the dehalogenation step catalyzed by dehaloperoxidase (DHP) from Amphitrite ornata, an unusual heme-containing protein with a globin fold and peroxidase activity, has remarkable similarity with that of the classical heme peroxidase, horseradish peroxidase (HRP). Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, we have concluded that two sequential one electron oxidations of the halogenated phenol substrate leads to a cationic intermediate that strongly resembles a Meisenheimer intermediate – a commonly formed reactive complex during nucleophilic aromatic substitution reactions especially in the case of arenes carrying electron withdrawing groups.}, number={1}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, author={Szatkowski, Lukasz and Thompson, Matthew K. and Kaminski, Rafal and Franzen, Stefan and Dybala-Defratyka, Agnieszka}, year={2011}, month={Jan}, pages={22–32} }
 @article{thompson_franzen_ghiladi_reeder_svistunenko_2010, title={Compound ES of Dehaloperoxidase Decays via Two Alternative Pathways Depending on the Conformation of the Distal Histidine}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja106620q}, DOI={10.1021/ja106620q}, abstractNote={Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that has been shown to catalyze the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2 treatment were studied by low-temperature electron paramagnetic resonance spectroscopy in the range of reaction times from 50 ms to 2 min at three different pH values. Two high-spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES (the enzyme with the heme in the oxoferryl state and a radical on the polypeptide chain) has been assigned to Tyr34, and the other radical has been assigned to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. High-performance liquid chromatography analysis of the reaction products showed a pH-dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH, formed under H2O2 in the absence of the trihalophenol substrates, is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH and formation of the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays, yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2 when no substrate is available.}, number={49}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Thompson, Matthew K. and Franzen, Stefan and Ghiladi, Reza A. and Reeder, Brandon J. and Svistunenko, Dimitri A.}, year={2010}, month={Dec}, pages={17501–17510} }
 @article{thompson_davis_serrano_nicoletti_howes_smulevich_franzen_2010, title={Internal Binding of Halogenated Phenols in Dehaloperoxidase-Hemoglobin Inhibits Peroxidase Function}, volume={99}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2010.05.041}, abstractNote={Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.}, number={5}, journal={BIOPHYSICAL JOURNAL}, author={Thompson, Matthew K. and Davis, Michael F. and Serrano, Vesna and Nicoletti, Francesco P. and Howes, Barry D. and Smulevich, Giulietta and Franzen, Stefan}, year={2010}, month={Sep}, pages={1586–1595} }
 @article{ma_thompson_gaff_franzen_2010, title={Kinetic Analysis of a Naturally Occurring Bioremediation Enzyme: Dehaloperoxidase-Hemoglobin from Amphitrite ornata}, volume={114}, ISSN={["1520-6106"]}, DOI={10.1021/jp1014516}, abstractNote={The temperature dependence of the rate constant for substrate oxidation by the dehaloperoxidase-hemoglobin (DHP) of Amphitrite ornata has been measured from 278 to 308 K. The rate constant is observed to increase over this range by approximately a factor of 2 for each 10 °C temperature increment. An analysis of the initial rates using a phenomenological approach that expresses the peroxidase ping-pong mechanism in the form of the Michaelis-Menten equation leads to an interpretation of the effects in terms of the fundamental rate constants. The analysis of kinetic data considers a combination of diffusion rate constants for substrate and H(2)O(2), elementary steps involving activation and heterolysis of the O-O bond of H(2)O(2), and two electron transfers from the substrate to the iron. To complete the analysis from the perspective of turnover of substrate into product, density function theory (DFT) calculations were used to address the fate of phenoxy radical intermediates. The analysis suggests a dominant role for diffusion in the kinetics of DHP.}, number={43}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Ma, Huan and Thompson, Matthew K. and Gaff, John and Franzen, Stefan}, year={2010}, month={Nov}, pages={13823–13829} }
 @article{nicoletti_thompson_howes_franzen_smulevich_2010, title={New Insights into the Role of Distal Histidine Flexibility in Ligand Stabilization of Dehaloperoxidase-Hemoglobin from Amphitrite ornata}, volume={49}, ISSN={["0006-2960"]}, DOI={10.1021/bi9020567}, abstractNote={The present work highlights the important role played by the distal histidine in controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases. In DHP the distal histidine is highly mobile and undergoes a conformational change that places it within hydrogen-bonding distance of anionic ligands and water, where strong hydrogen bonding can occur. The detailed resonance Raman (RR) analysis at room temperature shows the presence of an equilibrium between a 5-coordinate and a 6-coordinate (aquo) high-spin form. The equilibrium shifts toward the aquo form at 12 K. These two forms are consistent with the existing X-ray structures where a closed conformation, with His55 positioned in the distal pocket and H-bonded with the distal water molecule (6-coordinate), and an open solvent-exposed conformation, with the His55 displaced from the distal pocket (5-coordinate form), are in equilibrium. Moreover, the comparison between the Raman data at 298 and 12 K and the results obtained by EPR of DHP in the presence of 4-iodophenol highlights the formation of a pure 5-coordinate high-spin form (open conformation). The data reported herein support the role of His55 in facilitating the interaction of substrate and inhibitor in the regulation of enzyme function, as previously suggested. The two conformations of His55 in equilibrium at room temperature provide a level of control that permits the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.}, number={9}, journal={BIOCHEMISTRY}, author={Nicoletti, Francesco P. and Thompson, Matthew K. and Howes, Barry D. and Franzen, Stefan and Smulevich, Giulietta}, year={2010}, month={Mar}, pages={1903–1912} }
 @article{d’antonio_d’antonio_thompson_bowden_franzen_smirnova_ghiladi_2010, title={Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata}, volume={49}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi100407v}, DOI={10.1021/bi100407v}, abstractNote={Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.}, number={31}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={D’Antonio, Jennifer and D’Antonio, Edward L. and Thompson, Matthew K. and Bowden, Edmond F. and Franzen, Stefan and Smirnova, Tatyana and Ghiladi, Reza A.}, year={2010}, month={Aug}, pages={6600–6616} }