Fluorescence study of the conformational properties of myoglobin structure

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Abstract
Tryptophanyl fluorescence of high-spin and low-spin complexes of sperm whale ferric- and ferrousmyoglobins, met-, azide- and cyanomyoglobins and deoxy-, oxy- and carboxymyoglobins has been studied in the pH range 2.5-13. The pH-dependent fluorescence of sperm whale metmyoglobin acylated at the N-terminal alpha-amino group by methylisothiocyanate and of bovine metmyoglobin, which contains invariant Trp7 and Trp14 but lacks Tyr151, have also been examined. Drastic changes in the fluorescence were registered in the acidic and alkaline pH ranges which are due to denaturation of Mb. Fluorescent and CD data indicate that at pH less than 4.5 and pH greater than 11.5 the unique spatial structure of the protein is destroyed whereas the secondary structure and integrity are essentially preserved. In all sperm whale and bovine myoglobins studied a local conformational change in the surroundings of Trp is observed which precedes alkaline denaturation. It seems to be due to deprotonation of lysine residues and breakage of the salt bridges essential for the maintenance of the native conformation of the N-terminal and the adjacent region. The parameters of this conformational transition are found to correlate with the spin state of the heme complex. However, analysis of the fluorescence behaviour of different ligand derivatives of myoglobin in the whole pH range studied enables one to conclude that the exact protein conformation depends not only on the spin state of the Fe atom but, to a greater extent, probably on the chemical nature of the ligand and its interaction with the protein groups in the heme cavity. Local conformational changes induced by the replacement of the sixth ligand or by varying pH seem to involve the same region of contacts between the A helix and GH fragment (or between the AE and GH helical complexes) though the extent of the changes may be different.