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...
The rates of oxygen uptake by rat liver mitochondria (MC) (native coupled, freshly frozen, and uncoupled by FCCP) have been measured polarographically in the absence (V(0)) or presence (V(1)) of 0.11-0.25 mM sperm whale MbO2. Under the same standard conditions, the rate of sperm whale MbO2 deoxygenation (V(2)) has been studied spectrophotometrically in the presence of respiring MC. For freshly frozen MC, the dependence of V(1) and V(2) on the overall charge of MbO2 has been investigated at pH 5.6-7.6, and the influence of other differently charged proteins (apomyoglobin, egg lysozyme, lactalbumin, and BSA) has been studied at pH 7.4. It is shown that the rate of mitochondrial respiration in the presence of MbO2 increases by 10-30% (V(1) > V(0)). No myoglobin effect is observed for FCCP-uncoupled MC (V(max) does not change). The rate of MbO2 deoxygenation is equal to the rate of oxygen uptake by mitochondria (V(2)/V(1) ~ 1 at pH 7.2-7.5). At varying pH < 7.2, the V(2) values become markedly higher than V(1), evidently due to the increased MbO2 positive charge and its stronger interaction with negatively charged mitochondrial membrane. At pH 7.4, on the contrary, V(2) is twice lower than V(1) in the case of negatively charged CM-MbO2 (pI 5.2), which has carboxymethylated histidines. Positively charged lysozyme (pI 11) strongly inhibits MbO2 deoxygenation (V(2)) without affecting oxygen uptake by MC (V(0) and V(1)). At the same time, apomyoglobin (pI 8.5), which is structurally very similar to the holoprotein, and both negatively charged lactalbumin (pI 4.4) and BSA (pI 4.7) have no substantial influence on V(2) and V(1). The MC membrane evidently has no specific sites for the interaction with myoglobin. Rather, the protein contacts with phospholipids of the outer membrane during MbO2 deoxygenation, and electrostatic interactions are of great importance for this process.
The spin label method developed by McConnell 15 years ago is now widely used in studies of the structure and dynamic properties of a variety of the biological systems such as proteins and protein complexes, lipids and membranes, nucleic acids, nucleoproteins, etc.The ESR spectrum of the nitroxide radcal – the spin label – is very sensitive to its microenvironment and permits easy registration of even subtle alterations in it. If spin labels are attached to different sites of a macromolecule the information can be gained about conformational properties of all these local regions and, as a result, about the dynamic behaviour of the object as a whole.
In this review, we shortly summarize the data of our studies (and also corresponding studies of other authors) on the new mechanism of myoglobin (Mb) deoxygenation in a cell, according to which Mb acts as an oxygen transporter, and its affinity for the ligand, like in other transporting proteins, is regulated by the interaction with the target, in our case, mitochondria (Mch). We firstly found that contrary to previously formulated and commonly accepted concepts, oxymyoglobin (MbO) deoxygenation occurs only via interaction of the protein with respiring mitochondria (low p values are necessary but not sufficient for this process to proceed). Detailed studies of the mechanism of Mb-Mch interaction by various physicochemical methods using natural and artificial bilayer phospholipid membranes showed that: (i) the rate of MbO deoxygenation in the presence of respiring Mch fully coincides with the rate of O2 uptake by mitochondria from a solution irrespectively of their state (native coupled, freshly frozen, or FCCP-uncoupled), i.e. it is determined by the respiratory activity of Mch; (ii) Mb nonspecifically binds to membrane phospholipids of the outer mitochondrial membrane, while any Mb-specific protein or phospholipid sites on it are lacking; (iii) oxygen uptake by Mch from a solution and the uptake of Mb-bound oxygen are two different processes, as their rates are differently affected by proteins (e.g. lysozyme) that compete with MbO for binding to the mitochondrial membrane; (iv) electrostatic forces significantly contribute to the Mb-membrane interactions; the dependence of these interactions on ionic strength is provided by the local electrostatic interactions between anionic groups of phospholipids (the heads) and invariant Lys and Arg residues near the Mb heme pocket; (v) interactions of Mb with phospholipid membranes promote conformational changes in the protein, primarily in its heme pocket, without significant alterations in the protein secondary and tertiary structures; and (vi) Mb-membrane interactions lead to decrease in the affinity of myoglobin for O2, which could be monitored by the increase in the MbO autooxidation rate under aerobic conditions and under anaerobic ones, by the shift in the MbO/Mb(2) equilibrium towards the ligand-free protein. The decrease in the affinity of Mb for the ligand should facilitate O2 dissociation from MbO at physiological p values in cells.
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