Interaction of iron(III) tetrasulphonated phthalocyanine with cytochrome c oxidase apoprotein

A complex of bovine cytochrome c oxidase protein with iron(III) tetrasulphonated phthalocyanine in place of hemes has been prepared. Its structure and properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, potentiometric measurements and polypeptide fragments examination. The visible absorption spectrum of Fe^III-apo-oxidase shows the main intense peak at 657 nm and weaker one at 700 nm. Molecular weight estimation demonstrated that one mole of the complex includes three functional units of MW 130,000 per unit. Spectroscopic examination of dithionite reduced Fe^III-apo-oxidase suggests the open crevice structure of the subunits containing iron tetrasulphonated phthalocyanine which is supported by the results of circular dichroism studies. Deep conformational changes of the protein upon displacement of hemes a and a₃ are not reversed upon Fe^IIIL⁺ incorporation into the protein. The molar ratio of the protein to Fe^IIIL in the complex (MW 130,000) was found to be 1:2. In the reduced form Fe^IIIL-apo-oxidase reacts with CN⁻, N^-₃ imidazole and molecular oxygen. Oxygen binding is irreversible, which indicates that the oxygen adduct is not of the oxyhemoglobin type. Electrophoretic and gel filtration studies of the SDS-urea dissociation products of cytochrome c oxidase and its phthalocyanine derivative suggest that Fe^IIIL and hemes a and a₃ are located on the same polypeptide fragments of the protein. Fe^IIIL-apo-oxidase is reduced by ferrous cytochrome c in agreement with their midpoint potentials which are 315.5 and 260 mV, respectively. However, the rate of the reaction of Fe^IIIL-apo-oxidase with ferrous cytochrome c is markedly lower than that of the native cytochrome c oxidase suggesting different mechanisms for this process in both cases.