Global Mapping of Structural Solutions Provided by the Extended X-ray Absorption Fine Structure ab Initio Code FEFF 6.01:  Structure of the Cryogenic Photoproduct of the Myoglobin−Carbon Monoxide Complex

Abstract

X-ray methods based on synchrotron technology have the promise of providing time-resolved structural data based on the high flux and brightness of the X-ray beams. One of the most closely examined problems in this area of time-resolved structure determination has been the examination of intermediates in ligand binding to myoglobin. Recent crystallographic experiments using synchrotron radiation have identified the protein tertiary and heme structural changes that occur upon photolysis of the myoglobin--carbon monoxide complex at cryogenic temperatures [Schlichting, I., Berendzen, J., Phillips, G., & Sweet, R. (1994) Nature 371, 808--812]. However, the precision of protein crystallographic data (approximately 0.2 A) is insufficient to provide precise metrical details of the iron--ligand bond lengths. Since bond length changes on this scale can trigger reactivity changes of several orders of magnitude, such detail is critical to a full understanding of metalloprotein structure--function relationships. Extended X-ray absorption fine structure (EXAFS) spectroscopy has the potential for analyzing bond distances to a precision of 0.02 A but is hampered by its relative insensitivity to the geometry of the backscattering atoms. Thus, it is often unable to provide a unique solution to the structure without ancillary structural information. We have developed a suite of computer programs that incorporate this ancillary structural information and compute the expected experimental spectra for a wide ranging series of Cartesian coordinate sets (global mapping). The programs systematically increment the distance of the metal to various coordinating ligands (along with their associated higher shells). Then, utilizing the ab initio EXAFS code FEFF 6.01, simulated spectra are generated and compared to the actual experimental spectra, and the differences are computed. Finally, the results for hundreds of simulations can be displayed (and compared) in a single plot. The power of this approach is demonstrated in the examination of high signal to noise EXAFS data from a photolyzed solution sample of the myoglobin--carbon monoxide complex at 10 K. Evaluation of these data using our global mapping procedures placed the iron to pyrrole nitrogen average distances close to the value for deoxymyoglobin (2.05 +/- 0.01 A), while the distance from iron to the proximal histidine nitrogen is seen to be 2.20 +/- 0.04 A. It is also shown that one cannot uniquely position the CO ligand on the basis of the EXAFS data alone, as a number of reasonable minima (from the perspective of the EXAFS) are observed. This provides a reasonable explanation for the multiplicity of solutions that have been previously reported. The results presented here are seen to be in complete agreement with the crystallographic results of Schlichting et al. (1994) within the respective errors of the two techniques; however, the extended X-ray absorption fine structure data allow the iron--ligand bond lengths to be precisely defined. An examination of the available spectroscopi...

Modulating carbon monoxide binding affinity and kinetics in myoglobin: the roles of the distal histidine and the heme pocket docking site

“…In all cases, the CO appears to be oriented with the C-O bond axis pointing toward the heme iron. A recent cryogenic EXAFS study of photolyzed MbCO [30] was found to be most consistent with the structure presented in Schlichting et al [17].…”

High-Throughput Computational and Experimental Techniques in Structural Genomics

“…The beamline configuration is similar to that used for focused beam X-ray absorption spectroscopy measurements (Chance et al 1996), with the monochromator set to 10 keV, and harmonic rejection using a Ni-coated mirror. The setup consists of a multiplate rail positioned at 45°with respect to the beam that brings a sample plate in a position close to the synchrotron X-ray source, an x-z stage and two ionization chambers placed before and after the sample plate for precise alignment of a sample in the plate to the beam, and a multi-element, fast count rate, highresolution Germanium detector placed perpendicular to the beam path, that captures the X-ray fluorescence.…”

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

“…The slightly lower edge energy of O 2 -␣Hb⅐AHSP compared with free O 2 -␣Hb suggests a lengthening of the Fe-O 2 bond in the AHSP complex. EXAFS data obtained from O 2 -␣Hb and O 2 -␣Hb⅐AHSP were fitted to models for the iron sites using restrained and constrained multiple-scattering models (58,(63)(64)(65). The observed and calculated EXAFS and the corresponding Fourier transforms are shown for O 2 -␣Hb and O 2 -␣Hb⅐AHSP in Fig.…”