Spectroscopic investigation of catalytic intermediates of the Chlorite Dismutase from Azospira oryzae: a treasure hunt

Anthropogenic activities like chemical industries are releasing chlorinated species, such as perchlorate (ClO4-) or chlorite (ClO2-), into the environment. These substances are toxic and difficult to treat in the wastewater. However, nature offers a way to solve this problem. Perchlorate-respiring bacteria possess an enzyme, namely called Chlorite dismutase (Cld). It can detoxify chlorite (ClO2-) - a by-product of the perchlorate respiration - to chloride (Cl-) and molecular oxygen (O2). Therefore, it is interesting to characterise Clds in more detail to resolve its reaction mechanism and to investigate its potential for wastewater treatment.This work presents new and direct evidence for transient high valent heme species during the reaction mechanism of an interesting enzyme, which can be applied for the bioremediation of chlorite.Life Science and Technology (LST

Health and Healthcare Utilization among Asylum-Seekers from Berlin’s LGBTIQ Shelter: Preliminary Results of a Survey

Gottlieb ND, Püschmann C, Stenzinger F, et al. Health and Healthcare Utilization among Asylum-Seekers from Berlin’s LGBTIQ Shelter: Preliminary Results of a Survey. International Journal of Environmental Research and Public Health. 2020;17(12): 4514.Background: LGBTIQ asylum-seekers face multiple health risks. Yet, little is known about their healthcare needs. In 2016, Berlin opened the only major shelter for LGBTIQ asylum-seekers in Germany. This preliminary study describes health and healthcare utilization by asylum-seekers living in Berlin’s LGBTIQ shelter. To identify particular healthcare needs, we compared our results to asylum-seekers from other shelters. Methods: We surveyed residents of the LGBTIQ shelter and 21 randomly selected shelters in Berlin, using a validated questionnaire in nine languages (n = 309 respondents, including 32 respondents from the LGBTIQ shelter). Bivariate tests and generalized linear mixed models were applied to examine differences in health and healthcare utilization between the two groups. Results: Residents of the LGBTIQ shelter show high rates of chronic and mental illness. They use ambulatory and mental health services more frequently than asylum-seekers from other shelters, including a significantly higher chance of obtaining psychotherapy/psychiatric care in case of need. Emergency room utilization is also higher in the LGBTIQ group. Conclusions: Asylum-seekers from the LGBTIQ shelter face high chronic and mental health burdens. Tailored services in the LGBTIQ shelter help obtain adequate healthcare; they should be scaled up to maximize their potential. Yet, unmet needs remain and warrant further research

Unique Biradical Intermediate in the Mechanism of the Heme Enzyme Chlorite Dismutase

Funding Information: This research was supported by the grant NWO–CW 711.014.006 from the Council for Chemical Sciences of The Netherlands Organization for Scientific Research. S.T. acknowledges the support from the Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE 2020─Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT─Fundação para a Ciência e a Tecnologia and from the European Union’s Horizon 2020 Research and Innovation Program through TIMB3 (grant agreements no 810856). This work is dedicated to the late Prof. Simon de Vries, who pioneered the development of the rapid kinetic techniques that have been seminal in this study. Publisher Copyright: © 2021 The Authors. Published by American Chemical Society.The heme enzyme chlorite dismutase (Cld) catalyzes O-O bond formation as part of the conversion of the toxic chlorite (ClO2-) to chloride (Cl-) and molecular oxygen (O2). Enzymatic O-O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae (AoCld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. AoCld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, AoCld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at kobs = 2-5 × 104 s-1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of AoCld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety's reorganization and thereby maximize the enzyme's catalytic efficiency.publishersversionpublishe

A traffic light enzyme: acetate binding reversibly switches chlorite dismutase from a red- to a green-colored heme protein

Chlorite dismutase is a unique heme enzyme that catalyzes the conversion of chlorite to chloride and molecular oxygen. The enzyme is highly specific for chlorite but has been known to bind several anionic and neutral ligands to the heme iron. In a pH study, the enzyme changed color from red to green in acetate buffer pH 5.0. The cause of this color change was uncovered using UV-visible and EPR spectroscopy. Chlorite dismutase in the presence of acetate showed a change of the UV-visible spectrum: a redshift and hyperchromicity of the Soret band from 391 to 404 nm and a blueshift of the charge transfer band CT1 from 647 to 626 nm. Equilibrium binding titrations with acetate resulted in a dissociation constant of circa 20 mM at pH 5.0 and 5.8. EPR spectroscopy showed that the acetate bound form of the enzyme remained high spin S = 5/2, however with an apparent change of the rhombicity and line broadening of the spectrum. Mutagenesis of the proximal arginine Arg183 to alanine resulted in the loss of the ability to bind acetate. Acetate was discovered as a novel ligand to chlorite dismutase, with evidence of direct binding to the heme iron. The green color is caused by a blueshift of the CT1 band that is characteristic of the high spin ferric state of the enzyme. Any weak field ligand that binds directly to the heme center may show the red to green color change, as was indeed the case for fluoride