SPring-8, the large synchrotron radiation facility

(a) Cytochrome c oxidase is a proton pump powered by the free energy derived from the reduction of oxygen from the air at the active site^[6] (red circle), thus a concentration gradient of proton is formed across the biological membrane. The concentration gradient of proton is indispensable for the biosynthesis of adenosine triphosphate as the energy substance for life. The reduction of oxygen to water and the proton pumping across the mitochondrial membrane are schematically shown by the light green and orange arrows, respectively. The right image is a magnified view of the radiation-damage-free active site with electron density observed at SACLA in this research. As shown by the white arrow, a peroxide anion formed from oxygen binds to two metal ions (Cu_B and Fea₃ of heme a₃) that form the active site.
(b) In conventional X-ray crystallography, peroxide anions are broken by X-ray irradiation, resulting in an increase in the apparent bond length corresponding to the electron density of peroxide anions (shown in the blue ellipse). Thus, the structure of the active site before X-ray irradiation is not accurately determined by conventional X-ray crystallography.

(a) Setup for taking X-ray diffraction images of radiation-damage-free protein crystals using X-ray laser at SACLA. The target position of a crystal is accurately irradiated with an X-ray laser.
(b) In the femtosecond crystallography, an X-ray diffraction image is obtained by irradiating a 10 fs X-ray laser pulse. To avoid the effect of radiation damage by the previous irradiations, the crystal is translated before every X-ray pulse irradiation to record X-ray diffraction images from a fresh portion of the crystal. The crystal is also rotated by a certain degree between X-ray irradiations to obtain three-dimensional X-ray diffraction images. Conventional X-ray crystallography requires a long exposure time (several seconds), causing proteins to undergo radiation damage.

(a) To examine the interatomic distance between oxygen atoms of the peroxide anion that most clearly represents the electron density shown by the red arrow in (b), structure refinement calculations were carried out for peroxide anion models different in their interatomic distances. The residual electron density around the peroxide anion model (the navy-blue cage models) was lowest for a distance of 1.55 Å between oxygen atoms, meaning that the model with this interatomic distance is suitable. The changes in the residual electron density owing to the difference in the peroxide anion model used for calculation are shown by the two green lines.
(b) Radiation damage-free electron density map of oxygen-reducing site. The red arrow indicates the electron density of the peroxide anion that binds to two metal ions (red sphere, Fe ion; blue sphere, Cu ion). The almost comparable peak heights of the electron densities at position A of the peroxide-anion-bound and the cyanide-bound crystals clearly showed that the data collected by the femtosecond crystallography is free of radiation damage. The actual peak heights are shown in panel c. Water molecules generated as a result of radiation damage of peroxide are trapped by adjacent tyrosine residues (electron density shown by A). The cyanide does not form water molecules upon X-ray irradiation, therefore, the peak height of the peroxide-bound crystal comparable to the cyanide-bound form means that radiation damage of the peroxide molecule did not occur during the data collection. The light blue spheres indicate the water molecules. Yellow bars indicate the bonds between carbon atoms. Yellow bars with blue bars indicate the bonds between nitrogen and carbon atoms and those with red bars indicate the bonds between oxygen and carbon atoms.
(c) Comparison of peak heights of the electron density A in (b) among the data of the peroxide-bound cytochrome c oxidase collected at SACLA (the femtosecond crystallography) and at SPring-8^[5] (the conventional crystallography) and the data of the cyanide-bound cytochrome c oxidase. The peak heights of the electron densities corresponding to A were normalized using the electron density around the water molecules, which remained unchanged upon X-ray irradiation.
(d) Radiation-damage-free structure of cytochrome c oxidase active site, corresponding to the site from Cu ion (Cu_B) to heme a₃ (Fea₃ and surrounding N atoms) in left figure of Fig. 1(b).