Mapping of yeast cytochrome c oxidase by fluorescence resonance energy transfer. Distances between subunit II, heme a, and cytochrome c bound to subunit III.

Fluorescence resonance energy transfer was used for measuring the distances between the following three sites of purified yeast cytochrome c oxidase: (a) a highly reactive sulfhydryl group on the mitochondrially made Subunit II; (b) endogenous heme a; (c) cytochrome c bound to the mitochondrially made Subunit III. Subunit II of purified cytochrome c oxidase was stoichiometrically and covalently labeled with the fluorophore N-(iodoacetamidoethyl)-l-aminonaphthalene-5-sulfonic acid or its 2,64somer without affecting enzymic activity. Similarly, a sulfhydryl group on Subunit III (which is situated at or near the cytochrome c binding site) was covalently labeled with thionitrobenzoateactivated yeast cytochrome c or its fluorescent porphyrin analog. The extent of resonance energy transfer between the various chromeand fluorophores was measured by steady state as well as nanosecond fluorimetry; the probable orientation factors of the various donor-acceptor pairs (which are essential for converting energy transfer efficiencies into actual distances) were estimated from nanosecond fluorescence depolarization measurements as well as from theoretical considerations. The following distances were obtained: Subunit II -+ heme a, 52 A; Subunit II + cytochrome c bound to Subunit III, 35 A; cytochrome c bound to Subunit III + heme a, 25 A. Electron transfer from the heme group of ferrocytochrome c to the heme a groups of cytochrome c oxidase must thus bridge a considerable distance. The quantitative information obtained in this study should contribute to an understanding of how cytochrome c oxidase functions and how its components are arranged within the mitochondrial inner membrane.