{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T22:27:02Z","timestamp":1762295222589,"version":"3.35.0"},"reference-count":44,"publisher":"Portland Press Ltd.","issue":"2","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2008,6,1]]},"abstract":"<jats:p>In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type\u00a01 copper site. The mutation of Leu386 to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile494 by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16\u00a0nm to 625\u00a0nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330\u00a0nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type\u00a01 as well as in the type\u00a02 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies &amp;lt;\u03bdCu-S&amp;gt;, calculated from resonance Raman spectra are 410\u00a0cm\u22121 for the wild-type enzyme and 396\u00a0cm\u22121 for the I494A mutant, indicating an increase of the Cu\u2013S bond length in the type\u00a01 copper site of the mutant. Overall the data clearly indicate that the Ile494 mutation causes a major alteration of the structure near the type\u00a01 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type\u00a01 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.<\/jats:p>","DOI":"10.1042\/bj20080166","type":"journal-article","created":{"date-parts":[[2008,5,9]],"date-time":"2008-05-09T11:01:07Z","timestamp":1210330867000},"page":"339-346","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":73,"title":["Proximal mutations at the type\u00a01 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants"],"prefix":"10.1042","volume":"412","author":[{"given":"Paulo","family":"Dur\u00e3o","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2781-901 Oeiras, Portugal"}]},{"given":"Zhenjia","family":"Chen","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2781-901 Oeiras, Portugal"}]},{"given":"Catarina\u00a0S.","family":"Silva","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2781-901 Oeiras, Portugal"}]},{"given":"Cl\u00e1udio\u00a0M.","family":"Soares","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2781-901 Oeiras, Portugal"}]},{"given":"Manuela\u00a0M.","family":"Pereira","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2781-901 Oeiras, Portugal"}]},{"given":"Smilja","family":"Todorovic","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2781-901 Oeiras, Portugal"}]},{"given":"Peter","family":"Hildebrandt","sequence":"additional","affiliation":[{"name":"Technische Universit\u00e4t Berlin, Institut f\u00fcr Chemie, Sekr. 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