Systems featuring a multi-copper oxidase associated with transition-metal complexes can be used to perform oxidation reactions in mild conditions. Here, a strategy is presented for achieving a controlled orientation of a ruthenium–polypyridyl graft at the surface of a fungal laccase. Laccase variants are engineered with unique surface-accessible lysine residues. Distinct ruthenium–polypyridyl-modified laccases are obtained by the reductive alkylation of lysine residues precisely located relative to the T1 copper centre of the enzyme. In none of these hybrids does the presence of the graft compromise the catalytic efficiency of the enzyme on the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Furthermore, the efficiency of the hybrids in olefin oxidation coupled to the light-driven reduction of O2 is highly dependent on the location of the graft at the enzyme surface. Simulated RuII–CuII electron coupling values and distances fit well the observed reactivity and could be used to guide future hybrid designs.

L.S. was the recipient of a MinistHre de l’Education Nationale fellowship. This study was supported by grants from the Agence Nationale de la Recherche (ANR-09-BLANC-0176 and ANR-15-CE07-0021-01) and from the Ministerio de EconomÍa, Industria y Competitividad (CTQ2016-79138-R). We thank Elise Courvoisier-Dezord from the Plateforme AVB (AMU): Analyse et Valorisation de la Biodiversit8 and Yolande Charmasson for help in the production of the recombinant enzymes, as well as Pascal Mansuelle and R8gine Lebrun from the Plateforme Prot8omique (CNRSAMU) for help in acquiring mass spectrometry data.

Peer Reviewed