@article{feducia_dumarieh_gilvey_smirnova_franzen_ghiladi_2009, title={Characterization of Dehaloperoxidase Compound ES and Its Reactivity with Trihalophenols†}, volume={48}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi801916j}, DOI={10.1021/bi801916j}, abstractNote={Dehaloperoxidase (DHP), the oxygen transport hemoglobin from the terebellid polychaete Amphitrite ornata, is the first globin identified to possess a biologically relevant peroxidase activity. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. Herein, we demonstrate that the first detectable intermediate following the addition of hydrogen peroxide to ferric DHP contains both a ferryl heme and a tyrosyl radical, analogous to Compound ES of cytochrome c peroxidase. Furthermore, we provide a detailed kinetic description for the reaction of preformed DHP Compound ES with the substrate 2,4,6-trichlorophenol and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a g ≈ 2.0058 signal confirming the presence of a protein radical in DHP Compound ES. In the absence of substrate, DHP Compound ES evolves to a new species, Compound RH, which is functionally unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between peroxidase and oxygen transport activities in this bifunctional protein.}, number={5}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Feducia, Jeremiah and Dumarieh, Rania and Gilvey, Lauren B. G. and Smirnova, Tatyana and Franzen, Stefan and Ghiladi, Reza A.}, year={2009}, month={Feb}, pages={995–1005} }
 @article{franzen_gilvey_belyea_2007, title={The pH dependence of the activity of dehaloperoxidase from Amphitrite ornata}, volume={1774}, ISSN={["1570-9639"]}, DOI={10.1016/j.bbapap.2006.09.019}, abstractNote={Dehaloperoxidase (DHP) from the terebellid polychaete, Amphitrite ornata, is the first hemoglobin that has peroxidase activity as part of its native function. The substrate 2,4,6-tribromophenol (TBP) is oxidatively debrominated by DHP to form 2,6-dibromoquinone (DBQ) in a two-electron process. There is a well-defined internal binding site for TBP above the heme, a feature not observed in other hemoglobins or peroxidases. A study of the pH dependence of the activity of DHP reveals a substantial difference in mechanism. From direct observation of the Soret band of the heme it is shown that the p K a for heme activation in protein DHP is 6.5. Below this pH the heme absorbance decreases in the presence of H 2 O 2 with or without addition of substrate. The low pH data are consistent with significant heme degradation. Above pH 6.5 addition of H 2 O 2 causes the heme to shift rapidly to a compound II spectrum and then slowly to an unidentified intermediate with an absorbance of 410 nm. However, the p K a of the substrate TBP is 6.8 and the greatest enzyme activity is observed above the p K a of TBP under conditions where the substrate is a phenolate anion (TPBO − ). Although the mechanisms may differ, the data show that both neutral TBP and anionic TPBO − are converted to the quinone product. The mechanistic implications of the pH dependence are discussed by comparison other known peroxidases, which oxidize substrates at the heme edge.}, number={1}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS}, author={Franzen, Stefan and Gilvey, Lauren B. and Belyea, Jennifer L.}, year={2007}, month={Jan}, pages={121–130} }
 @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} }
 @article{moses_brewer_lowe_lappi_gilvey_sauthier_tenent_feldheim_franzen_2004, title={Characterization of single- and double-stranded DNA on gold surfaces}, volume={20}, ISSN={["0743-7463"]}, DOI={10.1021/la0492815}, abstractNote={Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5‘-C6H12SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation−infrared reflection absorption spectroscopy (PM−IRRAS). The formation of dsDNA on the surface was identified by PM−IRRAS by a dsDNA IR signature at ∼1678 cm-1 that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 °C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au−S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.}, number={25}, journal={LANGMUIR}, author={Moses, S and Brewer, SH and Lowe, LB and Lappi, SE and Gilvey, LBG and Sauthier, M and Tenent, RC and Feldheim, DL and Franzen, S}, year={2004}, month={Dec}, pages={11134–11140} }