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Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 \u00c5 resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.<\/jats:p>","DOI":"10.3390\/molecules29020358","type":"journal-article","created":{"date-parts":[[2024,1,11]],"date-time":"2024-01-11T03:21:41Z","timestamp":1704943301000},"page":"358","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans"],"prefix":"10.3390","volume":"29","author":[{"given":"Kelly","family":"Frade","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4821-2358","authenticated-orcid":false,"given":"C\u00e9lia M.","family":"Silveira","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"given":"Bruno A.","family":"Salgueiro","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"given":"S\u00f3nia","family":"Mendes","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0082-9591","authenticated-orcid":false,"given":"L\u00edgia O.","family":"Martins","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"given":"Carlos","family":"Fraz\u00e3o","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"given":"Smilja","family":"Todorovic","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]},{"given":"Elin","family":"Moe","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.jbiotec.2016.08.011","article-title":"Bacterial enzymes involved in lignin degradation","volume":"236","author":"Colpa","year":"2016","journal-title":"J. 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