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abstracts
X-ray irradiation on a protein crystal can cause some subtle structural modification on the protein structure even if the radiation dose is much smaller than a dose used for a common crystal structure determination. In some case such structural modification increases ambiguity of structural inspection, and eventually could be an obstacle on the elucidation of structure basis of protein function. Bovine heart cytochrome c oxidase (CcO) is one of such proteins having some problem caused by the radiation damage. The proton pumping of CcO is coupled with O2 reduction at the O2 reduction site, thus accurate structure determination of bound ligand as well as CcO itself is very important. Whereas accurate structure determination was impeded by the immediate photolysis of the peroxide ligand upon X-ray irradiation even at a cryogenic temperature[1]. We developed a goniometer based data collection system for the femtosecond crystallography at SACLA (SPring-8 Angstrom Compact free-electron LAser). The femtosecond crystallography is expected to have an advantage in high-resolution and radiation damage free structure determination of very large protein by combined usage of large crystal and femtosecond intense X-ray pulse. In this presentation we are going to show the result of the femtosecond crystallography on the crystal of CcO having large unit cell dimensions. The close inspection of the electron density map calculated at 1.9 Å resolution showed the femtosecond crystallography worked fine for the high resolution and radiation damage free crystal structure determination of CcO.
abstracts
Bovine cytochrome c oxidase (CcO) pumps four protons in each catalytic cycle through H-pathway including a hydrogen-bond network and a water channel in tandem. Protons, transferred through the water-channel from the negative side of mitochondrial inner membrane into the hydrogen-bond network, are pumped to the positive side of the membrane electrostatically by net positive charges on a heme (heme a) iron created upon electron transfer to the O2 reduction site. For blockage of backward proton leak from the hydrogen-bond network, which determines the proton-pumping direction, the water channel is closed after O2 binding to initiate proton-pump. Thus, four protons must be collected in the hydrogen-bond network before O2 binding. The X-ray structural analyses of the oxidized/reduced CcO at 1.5/1.6 Å resolution reveal a large cluster composed of ~21 water molecules and a Mg2+ site including Glu198 (Subunit II) bridging CuA and Mg2+. The cluster of the oxidized state have 20 water sites with full occupancy and two sites with partial occupies of water, while that of the reduced state have 19 water sites with full occupancy and 3 sites with partial occupancies. The carboxyl group of Glu198 changes its coordination structure to Mg2+ upon the reduction of the active centers. The cluster is tightly sealed sterically against proton exchanges with the cluster outside except for a short hydrogen-bond network connecting the cluster with H-pathway. Five proton-acceptable groups hydrogen-bonded with the cluster suggest sufficient storage capacity for four proton equivalents. The redox-coupled structural changes in the electron transfer pathway from CuA, the initial electron acceptor from cytochrome c, to heme a suggest redox-driven effective proton donations from the cluster to H-pathway, facilitated by Glu198. These results indicate that the cluster is a crucial element of the proton-pumping system of bovine CcO.
