Evidences of Xenon-Induced Structural Changes in the Active Site of Cyano-MetMyoglobins: A 1H NMR Study

Using xenon atoms as a biomolecular probe raises the concern of whether they may influence in some way the molecular and electronic structure of the system under study. In this paper, the relevance of guest-host interactions in xenon complexes with paramagnetic myoglobins (Mbs) is thoroughly analyzed, and the issue about the use of xenon to detect and characterize voids within flexible biomolecules is critically discussed. A detailed 1H NMR study useful for describing the hydrophobic cavities close to the active site of low-spin ferric myoglobins with respect to their interaction with the xenon atom is presented. The method is subsequently validated by the analysis of Xe-Mb with two different myoglobins, extracted from horse and pig. These myoglobins differ by 14 amino acids. One of these, Ile142 in horse Mb, is located in the proximal cavity, which is the main xenon binding site in horse Mb, and is replaced by Met142 in pig Mb. We demonstrated specific behaviors associated with the capacity of each of the two myoglobins to bind xenon and provided site-specific information on the host-guest interaction. Moreover, 1H NMR measurements produce a picture of xenon-related local distortions of the protein, associated with a functionally relevant residue located right at the active site, the proximal hystidine E7(His93). According to the 1H NMR data, xenon induces the tilt of the residue His93 relative to the heme plane and consequently causes an alteration of the magnetic axes. Similar conclusions are obtained both for pig cyano-myoglobin and for horse cyano-myoglobin, the structural deformation being in the former of minor entity