Fluorescent monitoring of copper-occupancy in His-ended catalytic oligo-peptides.

Controlled generation of reactive oxygen species (ROS) is widely beneficial to various medical, environmental, and agricultural studies. As inspired by the functional motifs in natural proteins, our group has been engaged in development of catalytically active oligo-peptides as minimum-sized metalloenzymes for generation of superoxide anion, an active member of ROS. In such candidate molecules, catalytically active metal-binding minimal motif was determined to be X-X-H, where X can be most amino acids followed by His. Based on above knowledge, we have designed a series of minimal copper-binding peptides designated as G n H series peptides, which are composed of oligo-glycyl chains ended with C-terminal His residue such as GGGGGH sequence (G5H). In order to further study the role of copper binding to the peptidic catalysts sharing the X-X-H motif such as G5H-conjugated peptides, we should be able to score the occupancy of the peptide population by copper ion in the reaction mixture. Here, model peptides with Cu-binding affinity which show intrinsic fluorescence due to tyrosyl residue (Y) in the UV region (excitation at ca. 230 and 280 nm, and emission at ca. 320 nm) were synthesized to score the effect of copper occupancy. Synthesized peptides include GFP-derived fluorophore sequence, TFSYGVQ (designated as Gfp), and Gfp sequence fused to C-terminal G5H (Gfp-G5H). In addition, two Y-containing tri-peptides derived from natural GFP fluorophores, namely, TYG and SYG were fused to the G5H (TYG-G5H and SYG-G5H). Conjugation of metal-binding G5H sequence to GFP-fluorophore peptide enhanced the action of Cu(2+) on quenching of intrinsic fluorescence due to Y residue. Two other Y-containing peptides, TYG-G5H and SYG-G5H, also showed intrinsic fluorescence which is sensitive to addition of Cu(2+). There was linear relationship between the loading of Cu(2+) and the quenching of fluorescence in these peptide, suggesting that Cu(2+)-dependent quenching of Y-reside-derived fluorescence could be a measure of copper occupancy in the peptides. Lastly, the fate of Y residue in the Cu-loaded peptides under oxidative condition in the presence of H2O2 was discussed based on the Cu/H2O2-dependent changes in fluorescence spectra.

Production and removal of superoxide anion radical by artificial metalloenzymes and redox-active metals.

Generation of reactive oxygen species is useful for various medical, engineering and agricultural purposes. These include clinical modulation of immunological mechanism, enhanced degradation of organic compounds released to the environments, removal of microorganisms for the hygienic purpose, and agricultural pest control; both directly acting against pathogenic microorganisms and indirectly via stimulation of plant defense mechanism represented by systemic acquired resistance and hypersensitive response. By aiming to develop a novel classes of artificial redox-active biocatalysts involved in production and/or removal of superoxide anion radicals, recent attempts for understanding and modification of natural catalytic proteins and functional DNA sequences of mammalian and plant origins are covered in this review article.