This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objectives ?The objectives stated in the recent renewal proposal are these: We will improve our current single crystal UV/Vis microspectrophotometry facility to provide optical spectra from single crystals in an essentially seamless and routine fashion, at the X-ray beamline, will develop single crystal fluorescence spectroscopy, and will develop single crystal Raman spectroscopy. Results ?Our current capability at beamline X26-C consists of a Crystal Logic diffractometer with an ADSC Q4R area detector, a 4DX-ray Systems AB optical system, a Newport 75W Xe research arc lamp, and an Ocean Optics USB 4000 CCD-based spectrophotometer running SpectraSuite software on Windows XP or LINUX operating systems. We designed it to support routine collection of X-ray diffraction and optical absorption spectroscopic data. We have integrated both modes into the beamline control software in order to coordinate the data collection, and to link the results to our database tracking system, the PXDB. Visible light (350 - 850 nm) from a Xe arc lamp travels to the crystal, and then to the spectrophotometer, through quartz optical fibers. The 15x microscope objectives are based upon the Schwarzschild parabolic mirror design, which uses an all-reflecting principle and are, therefore, free from chromatic aberration. The light is focused to a spot size that depends upon objective and the diameter of the optical fiber to which it is connected. For example, the incident photons are focused to 25 ?m diameter spot through a 50 ?m optical fiber, whereas photons are collected through a 75 ?m diameter region focused by a 400 ?m optical fiber. This arrangement yields full range electronic absorption spectra typically in less than one second. The standard experiments involve the following steps, which are integrated into our beamline control software (CBASS): 1) Mount and center a crystal with the goniometer on the diffractomer. 2) Record 72 digital images of the loop/crystal, one every 5? around a full 360? rotation. 3) CBASS calls the C3D algorithm, which determines the broadest, flat face of the loop/crystal and directs the goniometer to rotate the crystal so that this face is presented to the incident objective lens for spectroscopy. This establishes the """"""""best"""""""" spectroscopy angle for the cryoloop and the crystal. It also avoids the cryoloop, which introduces artifacts if it were to intersect with the spectroscopy photon path. 4) Because macromolecular crystals often yield anisotropic optical spectra, several spectra are collected as a function of rotation angle;for example, best angle ? 30? in 5? or 10? increments, and another set centered at the best angle plus 180?. From this point forward, two mutually exclusive types of data can be collected: time-resolved X-ray dose dependent (Step 5) or diffraction data-dependent (skip to Steps 6 - 7). 5) Optical spectra between 350 - 850 nm are collected from a stationary crystal, in the best orientation, in a time-dependent mode, during which the X-ray shutter is opened at a selected time and for a designated exposure time. This provides a data set from which reaction kinetics can be determined from the analysis of changes in optical spectra at one or more wavelengths. 6) Following best practices, the researcher screens the crystal for X-ray diffraction, indexes the unit cell, and determines the data collection strategy. After CBASS queries the user for input regarding the desired intervals to collect optical spectra, it starts the X-ray diffraction data collection. 7) At the preselected intervals CBASS rotates the crystal back to the best orientation and collects another optical spectrum. This occurs during the readout of the Q4 X-ray detector and while the X-ray shutter is closed. CBASS then continues executing the data collection. An example of a result from this facility is that obtained by Dr. Orville in collaboration with Drs. Gadda and Prabhakar. It was published in a report entitled, """"""""Crystallographic, Spectroscopic, and Computational Analysis of a Flavin C4a-Oxygen Adduct in Choline Oxidase,"""""""" that appeared recently in Biochemistry. Choline oxidase (CHO) from Arthrobacter globiformis is a FAD-dependent enzyme that catalyzes the two-step, four-electron oxidation of choline to glycine betaine, with betaine aldehyde as a two-electron oxidized intermediate. In the two oxidative half-reactions, two molecules of O2 are converted into two H2O2 molecules. We performed several spectroscopic measurements on a number of choline oxidase crystals including before and after X-ray exposure. Importantly, the difference spectra (after ?before) clearly shows a spectrum with ?max at 400 nm that is nearly identical to spectra obtained from flavin C4a-OOH or C4a-OH enzyme reaction intermediates. Typically these reactive oxygen intermediates exhibit half-lives of only several ms in solution, but remarkably, it is stable in the crystal at 100K. The time-dependent results show that this species is generated very quickly upon X-ray exposure (Figure 2). The difference feature at 400 nm increases in an exponential process, and with a t1/2 of approximately 40 seconds. This rate is approximately commensurate with the decrease of the 460 and 485 nm features attributed to oxidized FAD with an apparent t1/2 of approximately 100 seconds. The resulting electron density maps (unbiased simulated annealing at 1.8 ? resolution) and the interpretation of the atomic structure is consistent with two possible reactive oxygen species, namely a covalent flavin C4a-OOH and/or C4a-OH adducts. Either structure correlates well with the spectroscopic observations. Moreover, each type of data reinforces the conclusions;whereas either type of data alone would yield somewhat ambiguous results. Plans ?Non-resonance and Resonance Raman Spectroscopy - Structure and function are undeniably linked. Since the Raman effect involves interactions between atomic positions, electron distribution, and intermolecular forces, it is ideally suited to provide insights into function that a crystal structure alone cannot. Indeed, the information available from Raman spectroscopy can be very detailed, exceeding the level of resolution found in all but the highest resolution X-ray crystal structures. It can also reveal changes in the distribution of electrons in a bound ligand as well as details about hydrogen bonding strengths between active site residues and substrate or inhibitor molecules. We have begun procurement of a micro-beam Raman instrument, which we hope to commission within the next half year. Off-line single-crystal spectroscopy ?We plan to construct a station for spectroscopy that duplicates the stations inside the x-ray hutch at beamline X26-C. Single Crystal, Fluorescence-Emission Spectroscopy ?Fluorescence spectroscopy complements the electronic absorption and Raman spectroscopy capabilities. Dr. Orville has just submitted a Challenge Grant to obtain apparatus to accomplish this at beamline X26-C, hoping to obtain apparatus for fluorescence spectroscopy at both the on-line and off-line single-crystal spectroscopy stations. Significance ?The technology we are developing will be unique in the United States, and will be matched only by the equivalent system at the ESRF in France. The scientific problems that we and our users will address are central to the progress of macromolecular sciences in the United States. The national resource we envision will support unprecedented, highly correlated studies. The results will provide much needed data on the complex relationships among macromolecular atomic structure, electronic structure and chemistry. These data will be used by the large number of national and international researchers in the field. Our plans will place the United States in a leadership position in this area. Publications ?H?roux, A., Bozinovski, D.M., Valley, M.P., Fitzpatrick, P.F. and Orville, A.M. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction, Biochemistry 48, 3407-3416 (2009). Orville, A.M., Lountos, G.T., Finnegan, S., Gadda, G.,Prabhakar R. Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase, Biochemistry 48, 720-728 (2009).
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