Backbone dynamics of azurin in solution: slow conformational change associated with deprotonation of histidine 35.

15N relaxation measurements have been performed on the type Iota blue copper protein azurin from Pseudomonas aeruginosa. The relaxation times show that one loop (residues 103-108) and one turn (residues 74-77) display fast internal motions. The rest of the protein is rigid with an average order parameter S(2) of 0.85 +/- 0. 05. The copper binding site shows the same degree of rigidity even though is it composed of several loops and lies outside the beta-sheet sandwich. Substantial exchange broadening was found for a number of residues surrounding the side chain of His-35. The average exchange rate has been determined from NMR exchange spectroscopy experiments and is 45 +/- 6 s(-)(1) at 41 degrees C. The exchange broadening is caused by the protonation/deprotonation equilibrium of His-35. The NMR results indicate that the two structures of azurin observed by X-ray diffraction of crystals at pH 5.5 and 9.0 [Nar, H., Messerschmidt, A., Huber, R., Van de Kamp, M., Canters, G. W. (1991) J. Mol. Biol. 221, 765-772] are present in solution and that they interconvert slowly.