Fluorescence studies on the interaction of myoglobin with mitochondria.
To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO(2)) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6-8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO(2) and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO(2) and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6-8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K(m)) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K(m) on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K(m) values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia. gb_post@icb.psn.ru
G B
GB
Postnikova
E A
EA
Shekhovtsova
D020816:Amino Acid Motifs; D000818:Animals; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism*; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D014907:Whales
D009211:Myoglobin; C017595:oxymyoglobin