STRUCTURAL FEATURES AND THE REACTION-MECHANISM OF CYTOCHROME-OXIDASE - IRON AND COPPER X-RAY ABSORPTION FINE-STRUCTURE

X-ray edge absorption of Cu and extended fine structure studies of both Cu and Fe centers, were made of cytochrome oxidase from beef heart, Paracoccus denitrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme). The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the X-ray beam was controlled by low temperature (-140.degree. C) and was continuously monitored by simultaneous optical spectroscopy and EPR monitoring every 30 min of X-ray exposure. The structure of the active site, a cytochrome a3-Cu pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a S bridge with 3 ligands 2.60 .+-. 0.03 .ANG. from Fe.alpha.3 and 2.18 .+-. 0.03 .ANG. from Cu.alpha.3. The distance between Fe.alpha.3 and Cu.alpha.3 is 3.75 .+-. 0.05 .ANG., making the S bond angle 103.degree. reasonable for sp3 S bonding. The Fe.alpha.3 first shell has 4 typical heme nitrogens (2.01 .+-. 0.03 .ANG.) with a proximal N at 2.14 .+-. 0.03 .ANG.. The 6th ligand is the bridging S. The Cu.alpha.3 first shell is identical to oxidized stellacyanin containing 2 N and a bridging S. Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cu.alpha.3 1st shell and contains the S that forms the bridge in fully oxidized and mixed valence formate states. The Fe.alpha.3 first shell is identical to HbO2 but has CO instead of O2. The other redox centers, Fe.alpha. and the other EPR detectable Cu are not observed in higher shells of Fe.alpha.3. Fe.alpha. has 6 equidistant N and Cua has 1 (or 2) N and 3 (or 2) S with typical distances; these ligands change only slight on reduction. These structures afford the basis for an oxygen reduction mechanism involving oxy- and peroxy intermediates.