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X-ray driven reduction of Cpd I of Catalase-3 from N. crassa reveals differential sensitivity of active sites and formation of ferrous state
- Zárate-Romero, Andrés, Stojanoff, Vivian, Cohen, Aina E., Hansberg, Wilhelm, Rudiño-Piñera, Enrique
- Archives of biochemistry and biophysics 2019 v.666 pp. 107-115
- Neurospora crassa, X-radiation, active sites, bioremediation, catalase, crystals, data collection, food technology, hydrogen peroxide, medicine, oxidation
- Catalases are biotechnologically relevant enzymes because of their applications in food technology, bioremediation, and biomedicine. The dismutation of hydrogen peroxide occurs in two steps; in the first one, the enzyme forms an oxidized compound I (Cpd I) and in the second one, the enzyme is reduced to the ferric state. In this research work, we analyzed the reduction of Cpd I by X-ray radiation damage during diffraction experiments in crystals of CAT-3, a Large-Size Subunit Catalase (LSC) from Neurospora crassa. A Multi-Crystal Data collection Strategy was applied in order to obtain the Cpd I structure at a resolution of 2.2 Å; this intermediate was highly sensitive to X-ray and was easily reduced at very low deposited radiation dose, causing breakage of the Fe=O bond. The comparison of the structures showed reduced intermediates and also evidenced the differential sensitivity per monomer. The resting ferric state was reduced to the ferrous state, an intermediate without a previous report in LSC. The chemically obtained Cpd I and the X-ray reduced intermediates were identified by UV-visible microspectrometry coupled to data collection. The differential sensitivity and the formation of a ferrous state are discussed, emphasizing the importance of the correct interpretation in the oxidation state of the iron heme.