HEMATOPORPHYRIN-PROMOTED PHOTOINACTIVATION OF MITOCHONDRIAL UBIQUINOL-CYTOCHROME-C REDUCTASE - SELECTIVE DESTRUCTION OF THE HISTIDINE LIGANDS OF THE IRON SULFUR CLUSTER AND PROTECTIVE EFFECT OF UBIQUINONE

Purified ubiquinol-cytochrome c reductase of beef heart mitochondria is very stable in aqueous solution; it suffers little damage upon illumination with visible light under aerobic or anaerobic conditions. However, it is rapidly inactivated when the photosensitizer hematoporphyrin is present during illumination. The hematoporphyrin-promoted photoactivation is dependent on sensitizer dose, illumination time, and oxygen. Singlet oxygen is shown to be the destructive agent in this system. The photoinactivation of ubiquinol-cytochrome c reductase is prevented by excess exogenous ubiquinone, regardless of its redox state. This protective effect is not due to protein-ubiquinone interactions but to the singlet oxygen scavenger property of ubiquinone. Ubiquinone also protects against hematoporphyrin-promoted photoinactivation of succinate-ubiquinone reductase and cytochrome c oxidase. The photoinactivation site in ubiquinol-cytochrome c reductase is the iron-sulfur cluster of Rieske's protein. Two histidine residues, presumably serving as two ligands for the iron-sulfur cluster of Rieske's protein, are destroyed. No polypeptide bond cleavage is detected. Photoinactivation has little effect on the spectral properties of cytochromes b and c1 but alters their reduction rates substantially. This photoinactivation also causes the formation of proton-leaking channels in the complex. When the photoinactivated reductase is co-inlaid with intact ubiquinol-cytochrome c reductase or cytochrome c oxidase in a phospholipid vesicle, no proton ejection can be detected during the oxidation of their corresponding substrates.