Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates
Atsuhiro Shimada, Fumiyoshi Hara, Kyoko Shinzawa‐Itoh, Nobuko Kanehisa, Eiki Yamashita, Kazumasa Muramoto, Tomitake Tsukihara, Shinya Yoshikawa
Abstract
Mammalian cytochrome c oxidase (CcO) reduces O 2 to water in a bimetallic site including Fe a3 and Cu B giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by singleelectron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a protonconducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at 1.8 resolution of fully reduced CcO crystals treated with O 2 for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained 45% of the O-form structure, 45% of the E-form structure, and 20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O-and E-forms exhibit an unusually long Cu B 2+ -OH -distance and Cu B