Azurin-Solvent interaction: an ESR spin labeling investigation

Abstract

We have investigated by means of electron spin resonance (ESR) spectroscopy using two spin labels, Iodoacetamido-proxyl and 3-Maleimido-proxyl, the dynamics of two different regions around the active site of azufin, a copper containing blue protein. The ESR measurements of spin labeled azufin have been carried out in the 110-300 K temperature range on wet (H20, D20 and ethanol/water mixtutes) and lyophilized samples. The behaviours of the outer hyperfme splitting separation, 2A', of the ESR spectra vs temperature of the lyophilized, and fully hydrated azurin in H20 and D20 suggest that the two spin labels ate located in regions of the protein surface with different dynamics and polarity. Moreover, all differences in the 2,4" values shown by the spin labeled azufin in normal and heavy water as well as the temperature behaviour disappear when azufin is in ethanol/water mixtares. The results ate discussed in temas of a close correlation between the molecular dynamics of the protein fragments to which the two spin labels are bound and the properties of the solvent used.