Unexpected photosensitivity of the well-characterized heme enzyme chlorite dismutase
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ORIGINAL PAPER
Unexpected photosensitivity of the well‑characterized heme enzyme chlorite dismutase Durga Mahor1 · Julia Püschmann1 · Diederik R. Adema1 · Marc J. F. Strampraad1 · Peter‑Leon Hagedoorn1 Received: 9 June 2020 / Accepted: 9 October 2020 © The Author(s) 2020
Abstract Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2− (chlorite) to innocuous Cl− and O2. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Graphic abstract Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between F eIII and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00775-020-01826-8) contains supplementary material, which is available to authorized users. Extended author information available on the last page of the article
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JBIC Journal of Biological Inorganic Chemistry
Keywords Chlorite dismutase · Azospira oryzae · Heme enzyme · Stopped-flow spectroscopy · UV–visible illumination · Photosensitivity · Electron paramagnetic resonance · Oligomeric state Abbreviations AoCld Azospira oryzae Chlorite dismutase Cld Chlorite dismutase CCld Cyanothece Sp. PCC7425 chlorite dismutase Cyt c Cytochrome c DaCld Dechloromonas aromatica Chlorite dismutase EPR Electron paramagnetic resonance eq. Equivalent HRP Horseradish peroxidase IPTG Isopropyl ß-d-1-thiogalactopyranoside KPi Potassium phosphate buffer SEC Size exclusion chromatography SF Stopped-flow SVD Singular value
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