Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a Tm of 47–51 C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with a-helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.This work was supported by grant BMC2002-01438 (Ministerio de Ciencia y Tecnología) and 9/UPV00042.310-13552 from Universidad del Pais Vasco. M.A.P. was supported by grant 01/1077 (Fondo de Investigación Sanitaria), grant BMC2002-00243 (Ministerio de Ciencia y Tecnología), and Red de Centros RCMN (C03/08).Peer reviewed