Studies relating to the formation of semi-met CuICuIIpanulirus interruptus haemocyanin
Abstract
Reduction of Panulirus interruptus haemocyanin in the methaemocyanin CuII2 state with N2H4 and S2O42– occurs in two stages. The product of the first stage is semi-met CuICuII 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 eaq– and CO2.– generated by pulse radiolysis occurs at some other sites on the protein, and no reduction of the CuII2 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 CuI2 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.