The pH-dependent fluorescence of intact sperm whale apomyoglobin (apo-Mb) containing two tryptophans at positions 7 and 14, and of apo-Mb derivatives modified on Trp7 by 2-hydroxy-5-nitrobenzyl bromide (Koshland reagent) and o-nitrophenylsulphenyl chloride, has been studied. The fluorescence of apomyoglobins modified at His residues by iodoacetamide and bromoacetate, and at the N-terminal cr-NH2 group by methylisothiocyanate, has also been investigated. The individual fluorescent properties of both tryptophans and their contributions to the total spectrum of apo-Mb have been resolved within the pH range 2-12.5. The quantum yield of the 'buried' Trpl4 (imax at 326 nm) is shown to be twofold higher at pH >8.5 than that of the 'exposed' Trp7 (A, , , at 333 nm). At pH 8.5-5.5 the fluorescence of Trpl4 diminished approximately twofold due to quenching by the ionized His residue, most probably His1 19. The quenching is evidently dynamic because the fluorescence lifetime is shown to be linearly proportional to quantum yield in this pH range. The fluorescence of Trp7 practically does not change between pH 5.5 and 10.0 but increases 2.5-3-fold in the pH range 5.5-4.3 while the contribution of Trpl4 remains constant. The conformational changes at the N-terminal and in the region adjacent to it, as well as in the whole apo-Mb molecule in acidic, alkaline and neutral pH ranges, are considered. A relationship is revealed between conformational states of the heme crevice and the N-terminal part of apo-Mb.Knowledge of the conformational properties of a protein molecule is important for understanding the mechanism of its functioning. Studies of the conformational changes in monomeric and oligomeric globins induced by variations of pH of the medium and electron state of the heme are of particular interest because they allow one to elucidate the ways the ligand affinity is regulated.Conformational changes in the heme crevice and other parts of Mb-like proteins have been studied by many physical methods. In our work, fluorescence has been used to study pH-and ligand-induced conformational changes in myoglobins and related structures, apomyoglobin (apo-Mb) and a metal-free analog of Mb which is the complex of apo-Mb with protoporphyrin IX. The structural changes at the Nterminal and in the adjacent region of the protein were followed by the fluorescence of two tryptophan residues localized in the A-helix, Trp7 (A5) and Trpl4 (A12). Those in the heme crevice were followed by emission of protophorphyrin IX Correspondence to G. B. Postnikova, Institute of Biological Physics of the USSR Academy of Sciences, 142292, Pushchino, Moscow Region, USSR.Abbreviations. met-Mb, metmyoglobin; CA-Mb, met-Mb alkylated at His residues by iodacetamide; CM-Mb, met-Mb alkylated at His residues by bromacetate; MITC-Mb, met-Mb acylated at the N-terminal by methylisothyocyanate (MITC); apo-Mb, myoglobin containing no heme; CA-apo-Mb, apo-Mb carboxyamidated on His; CM-apo-Mb, apo-Mb carboxymethylated on His; MITC-apo-Mb, apo-Mb modified by MITC; HNB-apo-Mb...
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 a-amino group by methylisothiocyanate and of bovine metmyoglobin, which contains invariant Trp7 and Trpl4 but lacks Tyrl51, 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 < 4.5 and pH > 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.The intrinsic fluorescence of heme proteins is known to be quenched due to efficient energy transfer to the prosthetic group [l, 21. However, it appears that this quenching is not complete, and it is possible to detect the fluorescence of native heme proteins. This was first demonstrated for sperm whale myoglobin (Mb) and lupine leghemoglobin (Lb) [3] and then for various hemoglobins (Hb) [4-71 by both highly sensitive right-angle optics [3, 4, 61 and front-face fluorometry [5, 71. The fluorescence quantum yield for Mb was found to be about 5% and for Hb about 1 % of that for Trp in solution.There is evidence [6-81 that the fluorescence of Hb is sensitive to ligand-induced changes in its quaternary structure, the so-called R-T transition. It is likely that conformational changes in the tertiary structure of monomeric globins induced by pH of the medium, electronic and ligand state of the heme can also be recorded by fluorescence [3]. In mammalian myoglobins, where both tryptophans are in the A-helix region adjacent to the N-terminal, this would enable one to establish a connection between the state of the heme and the conformation of the distant protein region.The aim of the prese...
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