Probing the environment of CuB in heme-copper oxidases

Abstract

Time-resolved step-scan FTIR (TRS2-FTIR) and density functional theory have been applied to probe the structural dynamics of CuB in heme-copper oxidases at room temperature. The TRS2-FTIR data of cbb3 from Pseudomonas stutzen indicate a small variation in the frequency of the transient CO bound to CuB in the pH/pD 7-9 range. This observation in conjunction with density functional theory calculations, in which significant frequency shifts of the v(CO) are observed upon deprotonation and/or detachment of the CuB ligands, demonstrates that the properties of the CUB ligands including the cross-linked tyrosine, in contrast to previous reports, remain unchanged in the pH 7-9 range. We attribute the small variations in the v(CO) of CUB to protein conformational changes in the vicinity of CuB. Consequently, the split of the heme Fe-CO vibrations (a-, β-, and γ-forms) is not due to changes in the ligation and/or protonation states of the CuB ligands or to the presence of one or more ionizable groups, as previously suggested, but the result of global protein conformational changes in the vicinity of Cu B which, in turn, affect the position of CuB with respect to the heme Fe. © 2007 American Chemical Society.