Structural investigation of pig metmyoglobin by 129Xe NMR spectroscopy

The potentiality of xenon's sensitivity to its local magnetic environment is thoroughly investigated to probe internal structural differences between pig and horse metmyoglobin (MMb). These MMb's differ by 14 amino acids. One of these, Ile142 in horse MMb, is located in the proximal cavity, which is the xenon-binding site in horse MMb, and is replaced by Met142 in pig MMb. Specific and non-specific xenon-protein interactions are investigated here by Xe-129 NMR chemical shifts and relaxation rate in aqueous solutions of pig MMb as a function of the xenon and protein concentrations. The results are complemented with Xe-129 NMR data of the isostructural carbonmonoxy myoglobin (COMb), with computational calculations in order to highlight the structural differences between the cavities, and H-1 NMR spectra to test the dependence of the H-1 chemical shift on the addition of xenon. The Xe-129 chemical shift NMR parameters are analysed quantitatively in terms of a two-site model. Xenon forms a 1:1 complex with the protein, characterized by an equilibrium binding constant K=[Xe](in)/([Xe](out)[MMb]), and exchanges rapidly between a cavity within the protein (Xe-in) and all other environments (Xe-out). A comparison of equilibrium constant, K (74 M-1) in pig and K (146 M-1) in horse, reveals differences in affinity of xenon to the interior of pig MMb. Changes in xenon binding in both pig and horse MMb are also pointed out by other experimental results, e.g. the difference in the estimated delta(in), which is shifted downfield in pig MMb and upfield in horse MMb, with respect to Xe-129 in buffer solution; the xenon-iron distance, 7.4 Angstrom, which is longer in the pig than was found in the horse, 5.3 Angstrom. (C) 2004 Elsevier B.V. All rights reserved.