Description
Our group has recently demonstrated, for the first time using a direct detection method, that a protein radical is formed in an enzyme of the group of peroxidases (located in Trp164 of the versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii). This type of radicals, which are formed by long-range electron transfer to the cofactor (heme group) activated by peroxide, seem to play a key role in the oxidative degradation of lignin and other recalcitrant compounds since: i) they are located at the protein surface, where they can enter into contact with the lignin macromolecule that is not allowed to interact directly with the heme group located at a central pocket with only a limited access; and ii) they form relatively stable free radicals with a strong oxidative power that expand the ability of the enzyme to oxidize recalcitrant substrates acting as redox mediators integrated in the protein molecule. In the RAPERO project the kinetic properties (including the transient states of the catalytic cycle) of native and modified peroxidases (obtained by site-directed mutagenesis) containing different amino-acid residues susceptible to form radicals involved in the oxidative catalysis of the enzyme, will be investigated. We will also investigate the structural determinants of lignin binding at the exposed radical environment, and their influence on the catalytic efficiency of the native enzymes and their mutated variants oxidizing different model substrates. Finally, the whole electron-transfer pathway from the protein surface to the heme group and its environment will be analyzed with respect to the formation and stability of the protein radical. Moreover, the existence of several catalytic sites in some of these enzymes, e.g. in the versatile peroxidase described for the first time at the CIB, will be considered to define the structure-function relationships responsible for their specialization in oxidation of different substrate types. The ligninolytic peroxidases do not act alone in lignin degradation, but they are part of a multienzymatic system including other oxidative enzymes providing the hydrogen peroxide required, acting on the degradation products or contributing to the initial attack to the polymer via diffusible chemical mediators forming stable radicals. Some of the above oxidoreductases present biotechnological interest for the development of industrial biocatalysts (as it is investigated in the European project NMP2-CT-2006-26456 coordinated by the CSIC with participation of 12 companies from different industrial sectors). Therefore, the structure-function studies planned in the present proposal will be of interest for the future development of improved oxidoreductases with industrial application.