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Biochemistry 2007, 46, 66-77
Manganese Oxidation Site in Pleurotus eryngii Versatile Peroxidase: A Site-Directed Mutagenesis, Kinetic, and Crystallographic Study†,‡ Francisco J. Ruiz-Duen˜as,§ Marı´a Morales,§ Marta Pe´rez-Boada,§ Thomas Choinowski,| Marı´a Jesu´s Martı´nez,§ Klaus Piontek,| and A Ä ngel T. Martı´nez*,§ Centro de InVestigaciones Biolo´ gicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain, and Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Schafmattstrasse 18 (HPM), ETH Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland ReceiVed July 31, 2006; ReVised Manuscript ReceiVed October 19, 2006
ABSTRACT: The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 Å) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (
