The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process.
Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 --> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.
Tsukihara
Tomitake
T
Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita 565-0871, Japan.
Shimokata
Kunitoshi
K
Katayama
Yukie
Y
Shimada
Hideo
H
Muramoto
Kazumasa
K
Aoyama
Hiroshi
H
Mochizuki
Masao
M
Shinzawa-Itoh
Kyoko
K
Yamashita
Eiki
E
Yao
Min
M
Ishimura
Yuzuru
Y
Yoshikawa
Shinya
S
D000818:Animals; D001224:Aspartic Acid; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D014961:X-Ray Diffraction
D021122:Protein Subunits; D011522:Protons; D001224:Aspartic Acid; D006418:Heme; D003576:Electron Transport Complex IV