X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obt...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences
Main Authors: Arcovito, Alessandro, Benfatto, Maurizio, Cianci, Michele, Hasnain, S. Samar, Nienhaus, Karin, Nienhaus, G. Ulrich, Savino, Carmelinda, Strange, Richard W., Vallone, Beatrice, Della Longa, Stefano
Format: Text
Language:English
Published: National Academy of Sciences 2007
Subjects:
Biological Sciences
Sperm whale
Online Access:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1851025
http://www.ncbi.nlm.nih.gov/pubmed/17404234
https://doi.org/10.1073/pnas.0608411104
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Summary:X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ±(0.02–0.07)-Å accuracy on the atomic distances and ±7° on the Fe–CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.

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