@article{moreno-chicano_carey_axford_beale_doak_duyvesteyn_ebrahim_henning_monteiro_myles_et al._2022, title={Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature}, volume={9}, ISSN={["2052-2525"]}, DOI={10.1107/S2052252522006418}, abstractNote={Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.}, journal={IUCRJ}, author={Moreno-Chicano, Tadeo and Carey, Leiah M. and Axford, Danny and Beale, John H. and Doak, R. Bruce and Duyvesteyn, Helen M. E. and Ebrahim, Ali and Henning, Robert W. and Monteiro, Diana C. F. and Myles, Dean A. and et al.}, year={2022}, month={Sep}, pages={610–624} }
 @article{mccombs_moreno-chicano_carey_franzen_hough_ghiladi_2017, title={Interaction of Azole-Based Environmental Pollutants with the Coelomic Hemoglobin from Amphitrite ornata: A Molecular Basis for Toxicity}, volume={56}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/acs.biochem.7b00041}, DOI={10.1021/acs.biochem.7b00041}, abstractNote={The toxicities of azole pollutants that have widespread agricultural and industrial uses are either poorly understood or unknown, particularly with respect to how infaunal organisms are impacted by this class of persistent organic pollutant. To identify a molecular basis by which azole compounds may have unforeseen toxicity on marine annelids, we examine here their impact on the multifunctional dehaloperoxidase (DHP) hemoglobin from the terebellid polychaete Amphitrite ornata. Ultraviolet-visible and resonance Raman spectroscopic studies showed an increase in the six-coordinate low-spin heme population in DHP isoenzyme B upon binding of imidazole, benzotriazole, and benzimidazole (Kd values of 52, 82, and 110 μM, respectively), suggestive of their direct binding to the heme-Fe. Accordingly, atomic-resolution X-ray crystal structures, supported by computational studies, of the DHP B complexes of benzotriazole (1.14 Å), benzimidazole (1.08 Å), imidazole (1.08 Å), and indazole (1.12 Å) revealed two ligand binding motifs, one with direct ligand binding to the heme-Fe, and another in which the ligand binds in the hydrophobic distal pocket without coordinating the heme-Fe. Taken together, the results demonstrate a new mechanism by which azole pollutants can potentially disrupt hemoglobin function, thereby improving our understanding of their impact on infaunal organisms in marine and aquatic environments.}, number={17}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={McCombs, Nikolette L. and Moreno-Chicano, Tadeo and Carey, Leiah M. and Franzen, Stefan and Hough, Michael A. and Ghiladi, Reza A.}, year={2017}, month={Apr}, pages={2294–2303} }
 @article{mccombs_d’antonio_barrios_carey_ghiladi_2016, title={Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin fromAmphitrite ornata}, volume={55}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/acs.biochem.6b00143}, DOI={10.1021/acs.biochem.6b00143}, abstractNote={The marine hemoglobin dehaloperoxidase (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of nitrophenols, an unprecedented nonmicrobial degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol (4-NP) as a representative substrate, the major monooxygenated product was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that the O atom incorporated was derived exclusively from H2O2, indicative of a peroxygenase mechanism for 4-NP oxidation. Accordingly, X-ray crystal structures of 4-NP (1.87 Å) and 4-NC (1.98 Å) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of persistent organic pollutants such as nitrophenols in the environment.}, number={17}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={McCombs, Nikolette L. and D’Antonio, Jennifer and Barrios, David A. and Carey, Leiah M. and Ghiladi, Reza A.}, year={2016}, month={Apr}, pages={2465–2478} }