Histidine at the active site of Neurospora tyrosinase

Abstract

The involvement of histidyl residues as potential ligands to the binuclear active-site Cu2+ of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Cu2+ measurements of photooxidized, reconsituted apoenzyme demonstrated the loss of binding of 1 Cu atom/mol of enzyme as a consequence of photosensitized oxidation of 3 out of 9 histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. Histodyl residues 188, 193 and 289 and may consitute metal ligands to 1 of the 2 active-site Cu atoms. Substitution of Cu by Co afforded complete protection of the histidyl residues from being modified by dye-sensitized photooxidation.