Distribution of copper atoms and binding of carbon monoxide in partially copper-depleted hemocyanin.
Binding of CO to single cuprous (half-apo) or cupric (met-apo) copper of hemocyanin is investigated by a new method which allows estimation of the total amount of CO bound to hemocyanin. Pure half-apo preparations could not be obtained with the molluscan hemocyanins from Helix pomatia and Octopus vulgaris, and a residual fraction of sites with coupled copper is always present. However, the determination of CO bound to the protein before and after addition of H2O2, used to oxidize selectively single copper sites, reveals that CO binds to half-apo Cu(I), and is released upon oxidation of copper to met-apo Cu(II). Binding of CO to half-apo is not associated to luminescence, proving that luminescence of native carboxyhemocyanin demands the presence of a second cuprous copper in the site. In addition, analysis of data indicates that the residual amount of coupled copper sites in partially copper-depleted hemocyanin is underestimated by the residual O2-copper band measured at 340 nm in air, while faithfully quantitated by the residual luminescence in the presence of CO. A distribution of the copper left in the site of three partially copper-depleted hemocyanins is depicted.
D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006372:Helix, Snails; D006433:Hemocyanins / Q000378:metabolism; D046911:Macromolecular Substances; D049831:Octopodiformes; D013057:Spectrum Analysis
D046911:Macromolecular Substances; D003300:Copper; D002248:Carbon Monoxide; D006433:Hemocyanins