Chapter 4 Collection of EXAFS data from oxidised and reduced Plastocyanin

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1 36 Chapter 4 Collection of EXAFS data from oxidised and reduced Plastocyanin 4.1 Preferred crystal orientations for collecting polarised EXAFS from poplar Pc Poplar Pc crystallises in the orthorhombic space group P As a result of the symmetry of this space group, data collected from a single crystal oriented with a unit-cell axis (a, b or c) parallel to the polarisation vector (ê) can be analysed as though it came from a single molecule. To simplify the analysis, all polarised EXAFS data were collected with the crystals oriented with ê//a, ê//b or ê//c. 4.2 Polarised EXAFS from oriented single crystals of oxidised Pc EXAFS data from oriented single crystals of Cu II Pc were collected in 1980 with an improvised system for orienting the crystal specimens (Scott et al., 1982). Rotation angles were optimised by inspecting prominent features in the X-ray absorption curves. While the crystal orientations were confirmed by X-ray precession photography after completion of the experiments, they may be inaccurate by as much as ±10 (H. C. Freeman, personal communication). The investigators were primarily interested to investigate the weak Cu-S δ (Met92) bond, which lies close to the c axis. The ê//c orientation maximising the contribution of this ligand to the EXAFS was attained by maximising the magnitude of a sharp pre-edge transition. Due to the morphology of the crystals, when this orientation was attained, the only other orientation easily attainable was that with ê//b. As a result, only data in these two orientations were collected. In retrospect, efforts should have been made to collect data with ê//a as well. To establish the (then unknown) reproducibility of the experiment, data were recorded from 3 crystal specimens. For 2 crystals ("crystal 2" and "crystal 7") data were recorded from both the ê//b and ê//c orientations. Only data in the ê//b orientation were recorded from the third crystal ("crystal 6").

2 37 For the present work, the averaged polarised Cu II Pc XAS data from crystals 2 and 7 were retrieved from the J. Am. Chem. Soc. data repository. The EXAFS was extracted using the program SPLINE (chapter 1; Ellis, 1995a). 4.3 Unpolarised EXAFS from frozen solutions of reduced Pc at ph 4.8 and 7.2 Lyophilised Cu II Pc (8.0 mg) was dissolved in 190 µl of a ph 7.2 buffer solution containing 0.1 M sodium phosphate, 0.05 M sodium ascorbate and 40% glycerol v/v. The pale pink solution was centrifuged and loaded into a lucite cell (outer dimensions mm 3, volume 180 µl) with an X-ray transparent front face of 60 µm Mylar tape. The final concentration of Pc was 4 mm. Lyophilised Cu II Pc (12.5 mg) was added to 200 µl of a ph 4.8 buffer solution containing 0.1 M sodium acetate, 0.05 M sodium ascorbate and 40% glycerol v/v. The mixture was centrifuged, separating a pale pink precipitate and a pale yellow supernatant. The supernatant was loaded into a lucite cell. The final concentration of Pc was estimated to be 2 mm. The loaded cells were plunged into liquid N 2. This converted each protein solution to a glass. The cells were stored under liquid N 2, and were later attached to the cryostat sample holder under liquid N 2. XAS data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) at the unfocussed 8-pole wiggler beam line 4-2 using a Si(220) double-crystal monochromator detuned 50% at 9643 ev. The storage ring current was between 25 and 49 ma. The samples were maintained at K in an Oxford Instruments CF1208 continuous-flow liquid He cryostat. The data were collected in fluorescence mode, using a Lytle detector with a Ni filter and Ar gas in the chamber. Standard N 2 -filled ion chambers were used to measure the incident intensity and the absorbance of a Cu-foil standard. As is frequently the case for Cu proteins, the Pc was contaminated with traces of Zn. This determined the upper limit of the energy as no useful data could be collected above the Zn K edge ( 9665 ev).

3 38 Data were collected between 8670 and 9643 ev. This energy range was divided into three regions. Large steps (10 ev) were used in the pre-edge region from 8670 to 8970 ev, small steps (0.35 ev) were used in the edge region from 8970 to 9020 ev to permit accurate energy calibration, and steps of increasing size ( ev) were used in the EXAFS region from 9020 to 9643 ev. No photooxidation of either sample was observed during data collection. When the samples were removed from the X-ray beam, the originally clear glass had turned brown where it had been exposed to the beam. 4.4 Polarised EXAFS from oriented single crystals of reduced Pc at ph 4.5 and 7.2 Crystals of Cu II Pc in contact with 2.99 M (NH 4 ) 2 SO 4 solution with 0.1 M sodium phosphate buffer at ph 6.0 were brought to ph's of 4.5 and 7.2 respectively by exchanging the solution with a series of solutions containing 2.99 M (NH 4 ) 2 SO 4 with mixed sodium phosphate/sodium acetate buffer (ph ) or sodium phosphate buffer (ph ). Each step was 0.2 ph units (final step for ph 4.5 was 0.1 units). The crystals were allowed to equilibrate for hours between changes. The final solutions contained 2.99 M (NH 4 ) 2 SO 4 with 0.1 M sodium acetate buffer at ph 4.5, and 2.99 M (NH 4 ) 2 SO 4 with 0.1 M sodium phosphate buffer at ph 7.2, respectively. The crystals were mounted in glass capillaries (wall thickness 0.01 mm, internal diameter 2 mm). Within the capillary, each crystal was reduced by adding a solution of 2.99 M (NH 4 ) 2 SO 4 and 0.05 M sodium ascorbate with 0.1 M sodium acetate buffer at ph 4.5 or 0.1 M sodium phosphate buffer at ph 7.2. When reduction was complete, excess solution was removed and the capillary sealed with wax. A drop of solution was left in the capillary away from the crystal to prevent dehydration. The capillaries were attached to brass pins with epoxy resin. Each brass pin was mounted in the top of a goniometer head. The orientation of each crystal was identified by X-ray precession photography and the crystal positioned in a known orientation using the goniometer head arcs. The crystal was transferred to the crystal orienter at the beam line and positioned with the appropriate unit cell axis parallel to the horizontal (polarisation) plane using the orienter χ and φ arcs (chapter 1).

4 39 XAS data were collected from the ph 4.5 crystals at the SSRL focussed wiggler beam line 6-2 using a Si(220) double-crystal monochromator detuned 55% at 9867 ev. The storage ring current was between 21 and 48 ma. The data were collected in fluorescence mode, using a Lytle detector with a Ni filter and Ar gas in the chamber. Standard N 2 -filled ion chambers were used to measure the incident intensity and the absorbance of a Cu-foil standard. XAS data were collected from the ph 7.2 crystals at the focussed wiggler beam line 4-2 using a Si(220) double-crystal monochromator detuned 0% at 9693 ev. The storage ring current was between 50 and 99 ma. The data were collected in fluorescence mode, using a liquid-n 2 cooled 13 element solid-state Ge array detector. In a typical scan, data were collected between 8670 ev and 9655 ev. This energy range was divided into three regions. Large steps (5.3 ev) were used in the pre-edge region from 8670 to 8970 ev, small steps (0.2 ev) were used in the edge region from 8970 to 9020 ev, and steps of increasing size ( ev) were used in the EXAFS region from 9020 to 9655 ev. The energy resolution of the monochromators were reduced by the use of focussing mirrors. The XAS spectra were broadened by 4 ev (half-width at half-height). Although this had little effect on the EXAFS, it is possible that small errors (estimated to be 0.2 ev) were introduced in the energy calibration. 4.5 Data reduction Using the program AVERAGE (Ellis, 1995b), the XAS data were energy-calibrated by means of the Cu-foil internal standard, the first inflection point on the Cu edge being assigned as ev, and the scans averaged using weights based on the signal/noise ratios. The averaged XAS data were edited to remove obvious monochromator glitches, and the EXAFS extracted as described previously using E 0 = ev for all data sets (chapter 1). The XAS data collection is summarised in table 4.1.

5 40 Table 4.1 Summary of XAS data collection from Pc. The ph of each sample was measured at room temperature. Time is the time taken to collect 1 scan. ph Temp. Crystal Crystal size (mm 3 ) Polarisation Detector Scans Time (min.) Oxidised Pc: Polarised EXAFS K ê // b 18 NaI ê // c " K ê // b " ê // c " Reduced Pc: Unpolarised EXAFS K Lytle K " Polarised EXAFS K ê // a Lytle ê // c " K ê // b " K ê // a 13 Ge K ê // b " K ê // c " element NaI scintillation detector.