NATURE AND FUNCTIONAL IMPLICATIONS OF THE CYTOCHROME-A3 TRANSIENTS AFTER PHOTODISSOCIATION OF CO-CYTOCHROME OXIDASE

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome alpha-3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (almost-equal-to 1-mu-s) as the post-photodissociation Cu(B)+-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from Cu(B) to Fe(a3) when CO binds to Cu(B), forming a cytochrome alpha-3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from Cu(B) binds on the distal side of cytochrome alpha-3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the Cu(B)/Fe(a3) pair. We speculate as to the identity of the ligand that is transferred between Cu(B) and Fe(a3) and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.