Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules

Postnikova, G. B.; Yumakova, Elena M.; Yakovleva, E. S.

doi:10.1111/j.1432-1033.1985.tb08892.x

Cited by 2 publications

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“…Earlier, using spin labels covalently bound to HisAlO in myoglobin [23] and to TyrE16 in leghemoglobin [24], we observed local conformational changes in the same pH range. They were interpreted as changes in the relative arrangement of the A helix and GH fragment (or AE and GH helical complexes).…”

Section: Conformational Changes In Myoglobin Structure: Effect Of P Hmentioning

confidence: 67%

Fluorescence study of the conformational properties of myoglobin structure

Postnikova¹,

Komarov²,

Yumakova³

1991

European Journal of Biochemistry

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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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Section: Conformational Changes In Myoglobin Structure: Effect Of P Hmentioning

confidence: 67%

Fluorescence study of the conformational properties of myoglobin structure

Postnikova¹,

Komarov²,

Yumakova³

1991

European Journal of Biochemistry

Self Cite

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show abstract

Fluorescence study of the conformational properties of myoglobin structure

Postnikova

Yumakova

1991

European Journal of Biochemistry

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The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5 -10.8 but change significantly at pH < 5.5 and pH > 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH < 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio 1325/1350 which diminishes at pH > 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintaince of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.In the previous parts of this work (see two preceding papers), we studied conformational changes in apomyoglobin and ferrous-and ferricmyoglobins at the N-terminal and in the region adjacent to it, by means of tryptophanyl fluorescence. In this paper, using the same technique, we have investigated pH-induced conformational changes both at the periphery of the myoglobin structure where both tryptophan residues, Trp7 (A5) and Trpl4 (A12), are localized, and at the 'active site' of myoglobin in the heme crevice. Since the ironporphyrin complex (heme) does not fluorescence, a metal-free analog of myoglobin, the complex of the apoprotein with protoporphyrin IX (PPIX-apo-Mb) was used which shows a strong emission in the visible spectral region. As has been shown earlier, PPIX readily enters the heme cavity of different heme proteins like myoglobin [l -31, hemoglobin [4-71, horseradish peroxidase [8] and cytochrome c [9].Correspondence to G. B. Postnikova, Institute of Biological Physics of the USSR Academy of Sciences, 142292, Pushchino, Moscow Region, USSR Ahhreviations. PPIX, protoporphyrin IX; PPIX-apo-Mb, complex of PPIX with apomyoglobin; MITC-apo-Mb, apomyoglobin modified by methyisothiocyanate (MITC); PPIX-apo-HRP, complex of PPIX with horseradish peroxidase; PPIX-apo-Hb, complex of PPIX with hemoglobin; apo-Mb, myoglobin con...

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Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules

Cited by 2 publications

References 6 publications

Fluorescence study of the conformational properties of myoglobin structure

Fluorescence study of the conformational properties of myoglobin structure

Fluorescence study of the conformational properties of myoglobin structure

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