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Abstract
Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3 Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba3-type and aa3-type cytochrome c oxidases around the proton-loading site are also described.
Highlights
Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane
The omit electron density map shows that the ligand bound at the active site in the oxidized form of ba[3] Cytochrome c oxidases (CcOs) is most likely a single-oxygen species corresponding to a water molecule or hydroxide ion bound between the heme a3 iron and CuB
Our conclusion is based upon the spherical shape of the electron density peak in the unbiased Fo-Fc omit map and comparison of residual densities when modelling a single water molecule at this site in other deposited CcO structures (Fig. 4)
Summary
Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Cytochrome c oxidases (CcOs) are the terminal enzymes of the respiratory chains in mitochondria and many bacteria They are integral membrane bound complexes that catalyse the reduction of molecular oxygen to water and utilize the energy released to translocate protons across the cell membrane. Cytochrome c delivers electrons from the positive side of the membrane at the same time as protons are taken up from the negative side of the membrane to the heme a3 - CuB active site, where the reaction with oxygen takes place. This creates a charge separation across the membrane which is further enhanced by the additional pumping of protons from the positive to the negative side of the membrane The key question is; what is the structural mechanism linking oxygen reduction to proton pumping?
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