JBIC (2000) 5 : 156–166 Q SBIC 2000 ORIGINAL ARTICLE Marco Giorgetti 7 Isabella Ascone 7 Mario Berrettoni Paolo Conti 7 Silvia Zamponi 7 Roberto Marassi In situ X-ray absorption spectroelectrochemical study of hydroxocobalamin Received: 26 July 1999 / Accepted: 22 October 1999 M. Giorgetti 7 I. Ascone (Y) Laboratoire pour l’Utilisation du Rayonnement Electromagnétique, Bâtiment 209 D, Centre Universitaire Paris-Sud, B.P. 34, F-91898 Orsay Cedex, France e-mail: ascone6lure.u-psud.fr Tel.: c33-1-64468092 Fax: c33-1-64464148 M. Berrettoni 7 P. Conti 7 S. Zamponi 7 R. Marassi Dipartimento di Scienze Chimiche, via S Agostino 1, I-62032 Camerino (MC), Italy Abstract An in situ X-ray absorption spectroscopy (XAS) spectroelectrochemical study of aquocobalamin (system B 12a -B 12r -B 12s ) has been carried out in aqueous solutions buffered at different pH values. To the best of our knowledge, this is the first structural study of aquo- cobalamin at room temperature under controlled oxi- dation conditions. Most of the previous work was in fact performed using frozen samples chemically treated to produce the species. The spectroelectrochemical ap- proach offers several advantages: (1) the reduction products may be studied without poisoning the system with chemical reductive reagents and (2) any possible variation of the oxidation state owing to the electrons produced by the incident beam is avoided as the elec- trode, under potentiostatic control, acts as a scavenger. The spectroelectrochemical approach, together with more careful data analysis, has led to an improved in- terpretation of the XAS data. These conditions were not met in previous works where the oxidation state was not controlled and multiple scattering contribu- tions were not taken into account. The general shape of the XAS spectra of the different species is not greatly affected by pH. A signature for the base-off square-pla- nar coordination has been evidenced for the Co(II) compound at basic pH. A new signature for Co(I), indi- cating square-planar coordination, has been identified on the experimental spectra and simulated in theoreti- cal X-ray absorption near-edge structure (XANES) studies. The flexibility of the electrochemical approach, that permits to unambiguously establish the formal oxi- dation state, has led to very reliable values for energy shift and peak intensity variations. The experimental XANES and extended X-ray absorption fine structure (EXAFS) spectra with a very good signal-to-noise ratio have been processed using the GNXAS package that takes into account multiple scattering contributions. EXAFS and XANES independent analysis result in the same structural model. The reduction from Co(III) to Co(II) produces the most significant structural changes: the cobalt coordination number decreases from six to five, and the edge position shifts by 2.4B0.3 eV. In ad- dition, the XANES spectra are strongly modified. The reduction from Co(II) to Co(I) produces mainly elec- tronic effects with no apparent change of the coordina- tion number. A discussion of the limits and potentiali- ties of EXAFS in this type of study has also been in- cluded. Key words X-ray absorption spectroscopy 7 Hydroxocobalamin 7 Spectroelectrochemistry 7 Oxidation state Introduction The different derivatives of vitamin B 12 [1, 2], or cobal- amins, are octahedral tetraaza macrocyclic complexes in which the central cobalt atom, formally in the c3 oxidation state, is bonded to the four nitrogens of the corrin ring. The nucleotide sidechain attached to a car- bon of the corrin ring terminates with a 5,6-dimethyl- benzimidazole group (DMB), that may act as axial li- gand (a) toward the cobalt atom. The other axial posi- tion (b) may be occupied by different coordination li- gands, including alkyl groups; cobalamin complexes in which the DMB is coordinated to the central cobalt atom are usually named base-on forms. Base-off forms are those in which the DMB is either not coordinated