A simple method to engineer a protein-derived redox cofactor for catalysis

Abstract

The 6x-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co2+. Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co2+-loaded 6xHis-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co2+-loaded 6xHis-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co2+-loaded 6xHis-tagged Cu1+ amicyanin oxidizes the copper site which is 20 Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.

Intra-electron transfer of amicyanin from newly derived active site to redox potential tuned type 1 copper site

“…A previous study illustrated the two step mechanism of H 2 O 2 interaction with Co 2? -loaded 69His-tagged site [1]. It is proposed that the initial step is the formation of a high-valent peroxocobalt (Co 3? )…”

“…Antibody drugs, fluorescence fused protein, and new enzymes with new or desirable functions are good examples of entities created by protein engineering. In a previous study, we tried to craft a protein to derive a new redox active cofactor for enzymatic catalysis and electron transfer [1]. The 69-Histidine tag was introduced into the N-terminus amicyanin ( Fig.…”

“…1A) mediating electron transfer from the methylamine dehydrogenase (MADH) to the cytochrome c-551i in P.denitrificans for bioenergetic processes [2][3][4]. Adding cobalt to a 69-Histidine tag, which is generally used for recombinant protein purification, plus H 2 O 2 mediates intra-electron transfer in amicyanin [1]. The idea is that the Co 2?…”