@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{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{serrano_davis_gaff_zhang_chen_d'antonio_bowden_rose_franzen_2010, title={X-ray structure of the metcyano form of dehaloperoxidase from Amphitrite ornata: Evidence for photoreductive dissociation of the iron-cyanide bond}, volume={66}, journal={Acta Crystallographica. Section D, Biological Crystallography}, author={Serrano, V. S. and Davis, M. F. and Gaff, J. F. and Zhang, Q. and Chen, Z. and D'Antonio, E. L. and Bowden, E. F. and Rose, R. and Franzen, S.}, year={2010}, pages={770–782} }
 @article{davis_gracz_vendeix_serrano_somasundaram_decatur_franzen_2009, title={Different Modes of Binding of Mono-, Di-, and Trihalogenated Phenols to the Hemoglobin Dehaloperoxidase from Amphitrite ornata}, volume={48}, ISSN={["0006-2960"]}, DOI={10.1021/bi801568s}, abstractNote={The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.}, number={10}, journal={BIOCHEMISTRY}, author={Davis, Michael F. and Gracz, Hanna and Vendeix, Franck A. P. and Serrano, Vesna and Somasundaram, Aswin and Decatur, Sean M. and Franzen, Stefan}, year={2009}, month={Mar}, pages={2164–2172} }
 @article{miksovska_horsa_davis_franzen_2008, title={Conformational Dynamics Associated with Photodissociation of CO from Dehaloperoxidase Studied Using Photoacoustic Calorimetry}, volume={47}, ISSN={["0006-2960"]}, DOI={10.1021/bi8012033}, abstractNote={Herein, we present photoacoustic calorimetry and transient absorption studies of the dynamics and energetics associated with dissociation of a ligand from Fe(2+) dehaloperoxidase (DHP) from Amphitrite ornata. Our data show that CO photodissociation is associated with an endothermic (DeltaH = 8 +/- 3 kcal mol(-1)) volume expansion (DeltaV = 9.4 +/- 0.6 mL mol(-1)) that occurs within 50 ns upon photodissociation. No additional thermodynamics were detected on slower time scales (up to 10 micros), suggesting that the dissociated ligand rapidly escapes from the heme-binding pocket into the surrounding solvent. Similar volume and enthalpy changes were observed for CO photodissociation in the presence of the substrate, 2,4-dichlorophenol or 4-bromophenol, indicating that either the substrate does not bind in the protein distal cavity at ambient temperature or its presence does not impact the thermodynamic profile associated with ligand dissociation. We attribute a fast ligand exchange between the protein active site and the surrounding solvent to the high flexibility of the distal histidine residue, His55, that provides a direct pathway between the heme-binding pocket and the protein exterior. The dynamics and energetics of conformational changes observed for dissociation of a ligand from DHP differ significantly from those measured previously for photodissociation of CO from the structural homologue myoglobin, suggesting that structural dynamics in DHP are fine-tuned to enhance the peroxidase function of this protein.}, number={44}, journal={BIOCHEMISTRY}, author={Miksovska, Jaroslava and Horsa, Simona and Davis, Michael F. and Franzen, Stefan}, year={2008}, month={Nov}, pages={11510–11517} }
 @article{nienhaus_nickel_davis_franzen_nienhaus_2008, title={Determinants of Substrate Internalization in the Distal Pocket of Dehaloperoxidase Hemoglobin of Amphitrite ornata}, volume={47}, ISSN={["0006-2960"]}, DOI={10.1021/bi801564r}, abstractNote={Dehaloperoxidase (DHP) is a small heme protein in the coelom of the terebellid polychaete Amphitrite ornata. It can act both as an oxygen storage protein (hemoglobin function) and as a dehaloperoxidase (peroxidase function). The X-ray structure of the ferric form shows that the phenolic substrate can bind inside the protein, which is not the case for a typical peroxidase. In the present study, we have used CO-ligated DHP to mimic the distal pocket of the peroxidase DHP and to probe under which conditions both a halophenol and a diatomic ligand can be accommodated in the distal pocket. To vary the structure of the distal pocket, we have compared wild-type DHP and mutants H55V and H55R at different pH values, using flash photolysis in the visible and FTIR spectroscopy in the CO stretching bands. The latter technique is extremely sensitive to even small structural changes in the CO environment and thus can report substrate binding in the distal pocket. Our results on wild-type DHP and its variants indicate that halophenols and a diatomic ligand can indeed simultaneously be present in the distal pocket if the distal histidine is in the low-pH conformation, in which its side chain is swung out of the distal pocket. The markedly different pH dependencies of enzyme activity and substrate binding are not consistent with the hypothesis that substrate dehalogenation occurs within the interior of DHP.}, number={49}, journal={BIOCHEMISTRY}, author={Nienhaus, Karin and Nickel, Elena and Davis, Michael F. and Franzen, Stefan and Nienhaus, G. Ulrich}, year={2008}, month={Dec}, pages={12985–12994} }
 @article{smirnova_weber_davis_franzen_2008, title={Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite ornata: HYSCORE experiments}, volume={130}, ISSN={["0002-7863"]}, DOI={10.1021/ja0772952}, abstractNote={Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is the first known hemoglobin to exhibit efficient peroxidase activity in the oxidation of phenolic substrates. Hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP was carried out to characterize effects of the substrate 2,4,6-trifluorophenol (TFP) binding on the iron coordination in order to elucidate the molecular mechanism of the change in protein function from a globin to a peroxidase. Continuous wave EPR spectra show that heme iron of DHP at pH 6.0 exists in the high spin state. HYSCORE spectra recorded at magnetic field corresponding to g = 2 revealed the presence of exchangeable protons with hyperfine coupling of ca. 6 MHz, consistent with a water molecule being the sixth ligand in the iron coordination. These protons' spectral features disappeared upon substrate binding. This observation highlights the proposed role of the substrate as a trigger for the switch from hemoglobin to peroxidase...}, number={7}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Smirnova, Tatyana I. and Weber, Ralph T. and Davis, Mike F. and Franzen, Stefan}, year={2008}, month={Feb}, pages={2128-+} }
 @article{serrano_chen_davis_franzen_2007, title={X-ray crystal structural analysis of the binding site in the ferric and oxyferrous forms of the recombinant heme dehaloperoxidase cloned from Amphitrite ornata}, volume={63}, ISSN={["2059-7983"]}, DOI={10.1107/S0907444907043417}, abstractNote={The dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is an enzyme that converts para-halogenated phenols to the corresponding quinones in the presence of hydrogen peroxide. Its enzymatic activity is similar to that of heme peroxidases such as horseradish peroxidase, yet it has the structural characteristics of the globin family of proteins, the main functions of which are oxygen transport and storage. In order to investigate the dual function of this hemoglobin peroxidase, the enzyme was expressed in Escherichia coli as a recombinant protein in its wild-type form and as a mutant protein in which Cys73 was replaced by a serine residue (C73S). Both the wild-type and mutant proteins were crystallized and their structures were determined at 100 K to a resolution of 1.62 A. The structure of the wild-type protein demonstrated that it was in the metaquo form, with the heme iron in the ferric oxidation state and the bound water lying 2.2 A from the heme iron. The structure of the C73S mutant protein was shown to contain a ferrous heme iron with a bound oxygen molecule. The bent bonding geometry of the Fe-O(1)-O(2) adduct results in a hydrogen bond of length 2.8 A between the second O atom, O(2), of molecular oxygen and N(2) of the distal histidine residue (His55) in both subunits contained within the asymmetric unit. This hydrogen-bonding interaction between His55 and the bound diatomic oxygen molecule provides new insight into the catalytic activation of H(2)O(2), which is essential for peroxidase activity.}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, author={Serrano, Vesna and Chen, Zuxu and Davis, Michael F. and Franzen, Stefan}, year={2007}, month={Oct}, pages={1094–1101} }
 @article{franzen_belyea_gilvey_davis_chaudhary_sit_lommel_2006, title={Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase}, volume={45}, ISSN={["0006-2960"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33746584767&partnerID=MN8TOARS}, DOI={10.1021/bi060020z}, abstractNote={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 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-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 H2O2. This observation suggests that the mechanism of DHP relies on H2O2 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.}, number={30}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Franzen, Stefan and Belyea, Jennifer and Gilvey, Lauren B. and Davis, Michael F. and Chaudhary, Chelsea E. and Sit, Tim L. and Lommel, Steven A.}, year={2006}, month={Aug}, pages={9085–9094} }
 @article{belyea_gilvey_davis_godek_sit_lommel_franzen_2005, title={Enzyme function of the globin dehaloperoxidase from Amphitrite ornata is activated by substrate binding}, volume={44}, ISSN={["0006-2960"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-28544440508&partnerID=MN8TOARS}, DOI={10.1021/bi051731k}, abstractNote={Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.}, number={48}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Belyea, J and Gilvey, LB and Davis, MF and Godek, M and Sit, TL and Lommel, SA and Franzen, S}, year={2005}, month={Dec}, pages={15637–15644} }
 @inproceedings{davis_wang_myers_iwamiya_kelley_1998, title={A study of the molecular interactions occurring in blends of cellulose esters and phenolic polymers}, booktitle={ACS Symposium Series 688, Cellulose Derivatives}, author={Davis, M. F. and Wang, X-M and Myers, M. D. and Iwamiya, J. H. and Kelley, S. S.}, editor={T. J. Heinze and Glasser, W. G.Editors}, year={1998}, pages={283} }