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A Quantitative XANES Analysis of the Calcium High-Affinity Binding Site of the Purple Membrane

Francesc Sepulcre ^*, M. Grazia Proietti , Maurizio Benfatto , Stefano Della Longa  ^¶, Joaquin García  and Esteve Padrós 

^* Departament d'Enginyeria Agroalimentària i Biotecnologia, Escola Superior d'Agricultura, Universitat Politècnica de Catalunya, Barcelona, Spain; Instituto de Ciencia de los Materiales de Aragón, CSIC-Universidad de Zaragoza, Facultad de Ciencias, Zaragoza, Spain; Laboratori Nazionali di Frascati-INFN, P. O. Box 13-00044, Frascati, Italy; ^¶ Dipartimento Medicina Sperimentale, University L'Aquila, L'Aquila, Italy; and Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain

Correspondence: Address reprint requests to Dr. Francesc Sepulcre Sanchez, Universitat Politècnica de Catalunya, Enginyeria Agroalimentària i Biotecnologia, Urgell 187, Barcelona, 08036 Spain. Tel.: 34-93-4137410; E-mail: francesc.sepulcre{at}upc.es.

In this article we report x-ray absorption measurements of Ca²⁺-substituted bacteriorhodopsin. We present a detailed study of the absorption spectrum close to the absorption edge that is very sensitive to the site geometry. We combined ab initio calculations of the x-ray absorption cross section based on a full multiple scattering approach, with a best fit of the experimental data performed by changing the cluster geometry. The Ca²⁺-bacteriorhodopsin environment is composed of six oxygen atoms showing a distorted orthorhombic symmetry, whereas the Ca²⁺ in water solution has a regular octahydrated first sphere of coordination. Our results are in good agreement with previous molecular models suggesting that the high-affinity cationic site could be in the proximity of the retinal pocket. Our results provide strong direct evidence of the specific binding site of the metal cation in bacteriorhodopsin.