Structural studies of mitochondrial coupling factor 1 using tyrosine fluorescence.
The absorbance and fluorescence spectral properties of mitochondrial F1-ATPase confirm that this protein does not contain tryptophan residues and therefore its fluorescence is due to tyrosines. The 36% increase in the fluorescence and the almost 100% increase in quantum yield upon denaturation of the protein suggest that a considerable number of tyrosyl residues have a very low quantum yield in the native enzyme. Quenching experiments using iodide indicate that all of the fluorophores are quenched and also all of them with the same quenching constant. These observations are interpreted as confirmatory of what has been found with several other proteins whose fluorescence originates from tyrosyl residues, where the buried tyrosines fluoresce with a much lower quantum yield than those which are exposed. ATP added to F1 previously depleted of loosely bound nucleotides changes the quenching constant of iodide and the quantum yield and this is interpreted to be due to a conformational change induced by the binding of the nucleotide to the enzyme. Addition of 2-mercaptoethanol decreases, although slightly, the polarization of the fluorescence. However, SDS addition gives a much bigger decrease. Hence disulphide bridges are less important for the tertiary structure of the protein than hydrophobic interactions, hydrogen bonding or other forces. Nevertheless the conformational change induced by reduction of disulphide bridges is detected in iodide quenching experiments and the change of the quantum yield of the enzyme.
Fernández-Belda
F
F
Teruel
J A
JA
Gómez-Fernández
J C
JC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D012974:Sodium Iodide / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000032:analysis*
D014443:Tyrosine; D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D012974:Sodium Iodide