Studies relating to the formation of semi-met CuICuIIpanulirus interruptus haemocyanin

Abstract

Reduction of Panulirus interruptus haemocyanin in the methaemocyanin Cu^II₂ state with N₂H₄ and S₂O₄^2– occurs in two stages. The product of the first stage is semi-met Cu^ICu^II which has a UV/VIS spectrum approximately midway between those of met- and deoxy-haemocyanin peaks λ/nm (ε/M^–1 cm^–1 per subunit) at 680 (≈120) and 337 (≈2900). At 25 °C (pH 8.7) with N₂H₄ as reduction rate constants k₁= 0.0268 M^–1 s^–1 and k₂ < 5 × 10^–4 M^–1 s^–1 have been obtained. Formation of semi-met (k₁) and deoxy-haemoyanin (k₂) at the intermediate and final stages is confirmed by reaction with O₂ to yield UV/VIS spectra of met- and oxy-haemocyanin respectively. Reduction of methaemocyanin with the strong reductants e_aq^– and CO₂^.– generated by pulse radiolysis occurs at some other sites on the protein, and no reduction of the Cu^II₂ site was observed. With the less strong reductant MV^.+ derived from methyl viologen (1,1′-dimethyl-4,4′-bipyridinium dichloride) and with methaemocyanin in excess, formation of semi-methaemocyanin was observed presumably by electron transfer from the protein surface. The same behaviour is noted with the photochemically generated 5-deazaflavin radical dfl^.– which does not further reduce the protein to the deoxyhaemocyanin state. With pulse-radiolysis-generated oxidising radicals N₃^. and (NCS)₂^.– deoxyhaemocyanin gives spectroscopically identifiable protein Tyr^.(410 nm) and Trp^.(510 nm) radicals, but no evidence for oxidation of the Cu^I₂ site. Mechanistic features are discussed including a consideration of the extent to which a redox partner can penetrate the protein to access the active site.