Tryptophan phosphorescence as a structural probe of mitochondrial F1-ATPase epsilon-subunit.
We report the detection of tryptophan phosphorescence emission from the sole residue in the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the epsilon-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the epsilon-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F1 in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads to a more compact structure of the epsilon-subunit, whereas the opposite occurs when Mg-ATP is added to nucleotide-depleted F1. These spectroscopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes involving the epsilon-subunit.
Solaini
G
G
Scuola Superiore di Studi Universitari e di Perfezionamento, S. Anna, Pisa, Italy.
Baracca
A
A
Parenti Castelli
G
G
Strambini
G B
GB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D003080:Cold Temperature; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008163:Luminescent Measurements; D008929:Mitochondria, Heart / Q000201:enzymology*; D009711:Nucleotides / Q000172:deficiency; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000187:drug effects; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D009711:Nucleotides; D014364:Tryptophan; D000255:Adenosine Triphosphate; D014508:Urea; D006180:Proton-Translocating ATPases; D019791:Guanidine