Naomine Yano

Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL

We report a method of femtosecond crystallography for solving radiation damage–free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-Å radiation damage–free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

The Mg2+-containing water cluster of mammalian cytochrome c oxidase collects four pumping proton equivalents in each catalytic cycle

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a proton-conducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H3O+ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O2 bound to heme a3. To block backward proton movement, the water channel remains closed after O2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg2+ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu198, which bridges the Mg2+ and CuA (the initial electron acceptor from cytochrome c) sites, suggest that the CuA-Glu198-Mg2+ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg2+-containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.

Distinguishing between Cl- and O2(2-) as the bridging element between Fe3+ and Cu2+ in resting-oxidized cytochrome c oxidase

Fully oxidized cytochrome c oxidase (CcO) under enzymatic turnover is capable of pumping protons, while fully oxidized CcO as isolated is not able to do so upon one-electron reduction. The functional difference is expected to be a consequence of structural differences: [Fe(3+)-OH(-)] under enzymatic turnover versus [Fe(3+)-O(2)(2-)-Cu(2+)] for the as-isolated CcO. However, the electron density for O(2)(2-) is equally assignable to Cl(-). An anomalous dispersion analysis was performed in order to conclusively demonstrate the absence of Cl(-) between the Fe(3+) and Cu(2+). Thus, the peroxide moiety receives electron equivalents from cytochrome c without affecting the oxidation states of the metal sites. The metal-site reduction is coupled to the proton pump.

Application of profile fitting method to neutron time-of-flight protein single crystal diffraction data collected at the iBIX

We developed and employed a profile fitting method for the peak integration of neutron time-of-flight diffraction data collected by the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC) for protein ribonuclease A and α-thrombin single crystals. In order to determine proper fitting functions, four asymmetric functions were evaluated using strong intensity peaks. A Gaussian convolved with two back-to-back exponentials was selected as the most suitable fitting function, and a profile fitting algorithm for the integration method was developed. The intensity and structure refinement data statistics of the profile fitting method were compared to those of the summation integration method. It was clearly demonstrated that the profile fitting method provides more accurate integrated intensities and model structures than the summation integration method at higher resolution shells. The integration component with the profile fitting method has already been implemented in the iBIX data processing software STARGazer and its user manual has been prepared.

X-ray structure of cyanide-bound bovine heart cytochrome c oxidase in the fully oxidized state at 2.0 Å resolution

The X-ray structure of cyanide-bound bovine heart cytochrome c oxidase in the fully oxidized state was determined at 2.0 Å resolution. The structure reveals that the peroxide that bridges the two metals in the fully oxidized state is replaced by a cyanide ion bound in a nearly symmetric end-on fashion without significantly changing the protein conformation outside the two metal sites.

Profile-fitting method to neutron time-of-flight protein single-crystal diffraction data collected at iBIX

iBIX is a time-of-flight neutron single-crystal diffractometer for elucidating mainly the hydrogen, protonation and hydration structures of biological macromolecules. iBIX is installed at BL03 at the Materials and Life Science Experimental Facility of J-PARC in Japan. The diffractometer was installed on a coupled moderator which has more intense peak and integrated intensity but more asymmetric and wider pulse shape than a decoupled and poisoned moderators. Intensities of the reflections from a protein crystal are relatively weak and some weak reflections are buried under the error of strong background by strong incoherent scattering of hydrogen atoms in protein crystals. Therefore, the methods to determine accurate integrated intensities of weak reflections are essential for protein neutron structural analysis. Thus, we attempted to find appropriate fitting function, develop profile fitting algorithm for integration method and apply it to full set neutron TOF protein single crystal diffraction data by using iBIX. As pulsed neutron shape is asymmetric, asymmetric fitting function must be used in profile fitting method. In order to determine proper fitting function, 4 asymmetric functions were evaluated using strong intensity peaks of neutron diffraction data from ribonuclease A collected at iBIX. It was shown that all 4 asymmetric functions fit well to strong intensity peaks and significant differences were not found. In order to reduce calculation time and the number of parameters, Gaussian convolved with two back-to-back exponentials was selected as a most suitable fitting function (Fig. 1). We developed test program and applied it to full set ribonuclease A and α-thrombin neutron diffraction data. Intensity statistics were calculated and joint refinements of

Current status and future prospects of single-crystal neutron diffractometer iBIX

Single crystal neutron diffraction is one of the powerful methods to obtain the structure information including the hydrogen atoms. IBARAKI biological crystal diffractometer called iBIX is a high performance time-of-flight single crystal neutron diffractometer to elucidate the hydrogen, protonation and hydration structures of organic compound and biological macromolecules in various life processes. iBIX is installed on the beam line No. 3 in Material and Life Science Facility, J-PARC. To realize high performance, we have succeeded to develop a new photon-counting two-dimensional detector system using scintillator sheets and wavelength-shifting fiber arrays for the X/Y axes. Since the end of 2008, iBIX has been available to user experiments supported by Ibaraki University. In 2012, we have upgraded the 14 existing detectors and install the 16 new detectors for diffractometer of iBIX. The total solid angle of detectors subtended by a sample and the average of detector efficiency become 2 and 3 times, respectively [1]. The total measurement efficiency of the present diffractometer was on one order of magnitude from the previous one coupled with the increasing of accelerator power. In the end of 2012, iBIX could be started to user experiments for biological macromolecules in earnest. In 2015, the accelerator power of J-PARC become 400~600kW. Achievement for full data set of biological macromolecules for neutron structure analysis by using iBIX is as follow. Maximum unit cell size was 132X132X132 angstrom in cube. Average sample volume was about 2~3mm in cube, Average measurement time was about 7~10 days. At the beginning of 2015, we distributed two press releases on scientific outcomes which make the most of the merit of the neutron diffraction experiment by iBIX [2]. Some interesting results obtained by iBIX will be reported in the presentation. In order to improve the quality of integrated intensity of weak reflections, we developed a profile fitting method for the peak integration of the data reduction software STARGazer. The profile fitting component was applied to the TOF diffraction data set of standard protein samples obtained by iBIX. From the results, the integrated intensities and model structure obtained by the profile fitting method were more accurate than those of summation integration method especially at higher resolution shells [3]. We already prepared its user manual and distribution package of the data reduction software including the profile fitting component. In the future, the accelerator power of J-PARC will be improved at 1MW. iBIX should be available regularly for full data set measurement of sample size of 1mm3. We will continue to develop the data reduction software and beam line instruments in order to improve the accuracy of intensity data obtained from small samples. We should successfully complete to develop the utility equipments for sample environments (heating, extension system for polymer sample, pulse laser system and cooling system for capillary enclosed sample). And then, those equipments will be started to apply for user experiment in 2017. We will report also the development status of iBIX in the presentation.

Intact structure determination of a highly radiation sensitive protein at SACLA

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.

Structures of bovine cytochrome oxidase reveal proton active transports

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.

Structural changes of bovine cytochrome c oxidase dependent on the redox states

Structural changes of bovine cytochrome c oxidase dependent on the redox states Kazumasa Muramoto,a,b Hidenori Fujisawa,a Naomine Yano,a Tomoko Maeda,a Kyoko Shinzawa-Itoh,a Eiki Yamashita,b Tomitake Tsukihara,b,c Shinya Yoshikawa,a,b aDepartment of Life Science and bPicobiology Institute, University of Hyogo, (Japan). cInstitute for Protein Research, Osaka University, (Japan). E-mail: muramoto@ sci.u-hyogo.ac.jp