@article{wu_zhao_franzen_tsai_2017, title={Bindings of NO, CO, and O-2 to multifunctional globin type dehaloperoxidase follow the 'sliding scale rule'}, volume={474}, journal={Biochemical Journal (London, England : 1984)}, author={Wu, G. and Zhao, J. and Franzen, S. and Tsai, A. L.}, year={2017}, pages={3485–3498} }
 @article{zhao_lu_franzen_2015, title={Distinct Enzyme-Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase}, volume={119}, ISSN={["1520-6106"]}, DOI={10.1021/acs.jpcb.5b07126}, abstractNote={The time-resolved kinetics of substrate oxidation and cosubstrate H2O2 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 kcat, Km(AH2) and Km(H2O2) 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.}, number={40}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Zhao, Jing and Lu, Chang and Franzen, Stefan}, year={2015}, month={Oct}, pages={12828–12837} }
 @article{zhao_moretto_le_franzen_2015, title={Measurement of Internal Substrate Binding in Dehaloperoxidase-Hemoglobin by Competition with the Heme-Fluoride Binding Equilibrium}, volume={119}, ISSN={["1520-5207"]}, DOI={10.1021/jp512996v}, abstractNote={The application of fluoride anion as a probe for investigating the internal substrate binding has been developed and applied to dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata. By applying the fluoride titration strategy using UV-vis spectroscopy, we have studied series of halogenated phenols, other substituted phenols, halogenated indoles, and several natural amino acids that bind internally (and noncovalently) in the distal binding pocket of the heme. This approach has identified 2,4-dibromophenol (2,4-DBP) as the tightest binding substrate discovered thus far, with approximately 20-fold tighter binding affinity than that of 4-bromophenol (4-BP), a known internally binding inhibitor in DHP. Combined with resonance Raman spectroscopy, we have confirmed that competitive binding equilibria exist between fluoride anion and internally bound molecules. We have further investigated the hydrogen bonding network of the active site of DHP that stabilizes the exogenous fluoride ligand. These measurements demonstrate a general method for determination of differences in substrate binding affinity based on detection of a competitive fluoride binding equilibrium. The significance of the binding that 2,4-dibromophenol binds more tightly than any other substrate is evident when the structural and mechanistic data are taken into consideration.}, number={7}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Zhao, Jing and Moretto, Justin and Le, Peter and Franzen, Stefan}, year={2015}, month={Feb}, pages={2827–2838} }
 @article{le_zhao_franzen_2014, title={Correlation of Heme Binding Affinity and Enzyme Kinetics of Dehaloperoxidase}, volume={53}, ISSN={["0006-2960"]}, DOI={10.1021/bi5005975}, abstractNote={Chemical and thermal denaturation of dehaloperoxidase-hemoglobin (DHP) was investigated to test the relative stability of isoforms DHP A and DHP B and the H55V mutant of DHP A with respect to heme loss. In thermal denaturation experiments, heme loss was observed at temperatures of 54, 46, and 61 °C in DHP A, DHP B, and H55V, respectively. Guanidinium hydrochloride (GdnHCl)- and urea-induced denaturation was observed at respective concentrations of 1.15 ± 0.01 M DHP A and 1.09 ± 0.02 M DHP B, and 5.19 ± 0.05 M DHP A and 4.12 ± 0.14 M DHP B, respectively. The binding affinity of heme appears to be significantly smaller in both isoforms of DHP than in myoglobins. This observation was corroborated by heme transfer experiments, in which heme was observed to transfer for DHP A and B to horse skeletal muscle myoglobin (HSMb). GdnHCl-induced denaturation suggests a threshold of 1 mM for stabilization by binding of the inhibitor 4-bromophenol (4-BP). Concentrations of 4-BP greater than 1 mM caused destabilization. Urea-induced denaturation showed only destabilizing effects from phenolic ligand binding. Heme transfer experiments from DHP to HSMb further support the hypothesis that the binding of halophenols to DHP facilitates the removal of the heme. Thermal denaturation assessed via UV-visible spectroscopy and that assessed by differential scanning calorimetry (DSC) are both in agreement with chemical denaturation experiments and show that the denaturing abilities of the halophenols improve with the size of the para halogen atom in 4-XP, where X = iodo, bromo, chloro, or fluoro (4-IP > 4-BP > 4-CP > 4-FP), and the number of halo substituents as in 2,4,6-tribromophenol (2,4,6-TBP > 4-BP). DHP B, which differs in five amino acids, is less stable than DHP A with ΔHcal and Tm values of 165.1 kJ/mol and 47.5 °C compared to values of 183.3 kJ/mol and 50.4 °C for DHP B and DHP A, respectively. Kinetic studies verified that DHP B has a catalytic efficiency (kcat/Km) ∼5-6 times greater than that of DHP A but showed an increased level of substrate inhibition in DHP B for both 2,4,6-TCP and 2,4,6-TBP. An inverse correlation between protein stability with respect to heme loss and catalytic efficiency is suggested on the basis of the fact that the heme in DHP B has a stability lower than that of DHP A but a catalytic efficiency higher than that of DHP A.}, number={44}, journal={BIOCHEMISTRY}, author={Le, Peter and Zhao, Jing and Franzen, Stefan}, year={2014}, month={Nov}, pages={6863–6877} }
 @article{barrios_d’antonio_mccombs_zhao_franzen_schmidt_sombers_ghiladi_2014, title={Peroxygenase and Oxidase Activities of Dehaloperoxidase-Hemoglobin from Amphitrite ornata}, volume={136}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja500293c}, DOI={10.1021/ja500293c}, abstractNote={The marine globin dehaloperoxidase‐hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2‐dependent oxidation of monohaloindoles, a previously unreported class of substrate for DHP. Using 5‐Br‐indole as a representative substrate, the major monooxygenated products were found to be 5‐Br‐2‐oxindole and 5‐Br‐3‐oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, 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 globin‐active oxyferrous form after peroxygenase activity ceases. Reactivity with 5‐Br‐3‐oxindole in the absence of H2O2 also yielded 5,5’‐Br2‐indigo above the expected reaction stoichiometry under aerobic conditions, and O2‐concentration studies demonstrated dioxygen consumption. Non‐enzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5’‐Br2‐indigo, and together suggest a novel oxidase activity for DHP.}, number={22}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Barrios, David A. and D’Antonio, Jennifer and McCombs, Nikolette L. and Zhao, Jing and Franzen, Stefan and Schmidt, Andreas C. and Sombers, Leslie A. and Ghiladi, Reza A.}, year={2014}, month={May}, pages={7914–7925} }
 @article{zhao_franzen_2013, title={Kinetic Study of the Inhibition Mechanism of Dehaloperoxidase-Hemoglobin A by 4-Bromophenol}, volume={117}, ISSN={["1520-6106"]}, DOI={10.1021/jp3116353}, abstractNote={The mechanism of dehaloperoxidase-hemoglobin (DHP) inhibition by 4-bromophenol (4-BP) was investigated using Michealis-Menten and transient-state kinetic analyses. Transient-state kinetics using the stopped-flow technique to mix DHP and H2O2 in the presence of inhibitor concentrations less than 10-fold greater than the enzyme concentration show that 4-BP does not fully impede H2O2 entering the distal pocket to activate DHP. It is not clear whether an oxoferryl intermediate is formed under these conditions and there may be alternative pathways for H2O2 reaction in the 4-BP bound form of DHP. Two new species have been identified during the reaction of 4-BP bound form of DHP in the transient-state kinetic experiment by using Singular Value Decomposition (SVD) and global-fitting analysis. Rather than forming Compound ES in the unbound form, an inhibitor bound intermediate that possesses blue-shifted Soret band and a double peaked Q-band is observed. This intermediate is subsequently converted to the end-point species that is distinguished from Compound RH formed in the uninhibited enzyme. Bench-top mixing kinetics of DHP were conducted in order to determine the inhibitor binding constant and to understand the enzyme inhibition mechanism from a thermodynamic perspective. It was found that the inhibition constant, Ki, decreased from 2.56 mM to 0.15 mM over the temperature range from 283 to 298 K, which permits determination of the enthalpy and entropy for inhibitor binding as -135.5 ± 20.9 kJ/mol and 526.1 ± 71.9 J/(mol·K), respectively, leading to the conclusion that inhibitor binding is entropically driven.}, number={28}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Zhao, Jing and Franzen, Stefan}, year={2013}, month={Jul}, pages={8301–8309} }
 @article{zhao_serrano_zhao_le_franzen_2013, title={Structural and Kinetic Study of an Internal Substrate Binding Site in Dehaloperoxidase-Hemoglobin A from Amphitrite ornata}, volume={52}, ISSN={["0006-2960"]}, DOI={10.1021/bi301307f}, abstractNote={X-ray crystal structures of dehaloperoxidase-hemoglobin 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 β- or 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 H2O 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.}, number={14}, journal={BIOCHEMISTRY}, author={Zhao, Jing and Serrano, Vesna and Zhao, Junjie and Le, Peter and Franzen, Stefan}, year={2013}, month={Apr}, pages={2427–2439} }