The binuclear cupric cluster of Cancer magister methemocyanin

Cancer magister hemocyanin oxidized by H₂O₂ (Felsenfeld, G.F. and Printz, M.P. (1959) J. Am. Chem. Soc. 81, 6259–6264) contained 80–95% cupric copper, small amounts of EPR-detectable Cu(II), and native hemocyanin. The small amounts of EPR-detectable Cu(II) showed a signal characteristic of mononuclear Cu(II) in the region of g = 2 between 233°K and 10°K, with normal Curie behavior, and no Δm = 2 signal. Magnetic susceptibility measurements show the methemocyanin to be diamagnetic over the temperature range 1.5–77°K. It had an optical absorption maximum at 680 nm, ϵ = 60 ± 7 M_Cu⁻¹ · cm⁻¹ at 25°C, and at 327 nm (ϵ = 3 · 10² M_Cu⁻¹ · cm⁻¹), 360 nm, 420 nm, and 680 nm at 77°K. CD bands were observed at 340, 400–450, and 650 nm (very broad). Methemocyanin was not reduced to O₂-binding cuprous hemocyanin by dithionite, hydroxylamine, ascorbate, ferrocyanide, H₂O₂, or superoxide. Based upon the normal Curie behavior of the small EPR-detectable signal, the absence of paramagnetism, and some similarities of optical spectra between methemocyanin and oxyhemocyanin, we conclude that the diagmagnetic Cu(II) of this methemocyanin occurs in spin-coupled binuclear Cu(II) clusters having a configuration related to the binuclear Cu(II) cluster in oxyhemocyanin; but that inability of methemocyanin to undergo reductive reactivation, and the low molecular extinction coefficients of the optical absorption bands indicate that some chemical or steric alteration, perhaps peroxidatic in nature, takes place during its formation.