X-ray absorption spectroscopy and electrochemistry on biological samples

In order to study metalloproteins, which change the metal oxidation stales during the catalytic cycle, we have developed an electrochemical cell for i n situ XAS measurement on biological samples. To be able to use proteins an d mutants that are usually available in small quantities the cell was desig ned to minimise: a) cavity of RVC working electrode and b) cavities for ele ctric contact between RVC working electrode and the other electrodes (count er and reference). The sample volume of 0.4 mi is sufficient for measuremen ts at several applied potentials. We have investigated the reduction of(a) the hydroxocobalamin (from Co(III) to Co(I)) and (b) microperoxidase (from Fe(III) to Fe(II)). We have then determined the correct energy shift of XAN ES in the two systems. In the case of hydroxocobalamin, reduction from Co(I II) to Co(II) produces the most significant structural changes (Giorgetti e t al. 1997) The reduction from Co(II) to Co(I) produces mainly electronic e ffects with no apparent change of the coordination number. Microperoxidase XANES spectrum shifts by 1 eV +/- 0.5 eV upon oxidation.