TRYPTOPHAN PHOSPHORESCENCE AS A STRUCTURAL PROBE OF MITOCHONDRIAL F1-ATPASE EPSILON-SUBUNIT

We report the detection of tryptophan phosphorescence emission from th e sole residue in the epsilon-subunit of the bovine heart mitochondria l 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, wi th the 0-0 vibrational band centered at 411 nm, reveals the hydrophobi c nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid so lution emphasize the existence of multiple conformations of the epsilo n-subunit, structures which are rather stable as they do not interconv ert in the millisecond time scale. Further, from the relatively long t riplet lifetime at 273 K, it is possible to infer the existence of a t ight, rigid core in the structure of the epsilon-subunit. Under subuni t-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all tem peratures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tr yptophanyl side-chain. This conclusion is supported by the results obt ained at 273 K by dissociating F1 in the presence of 0.3 M guanidine h ydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads t o a more compact structure of the epsilon-subunit, whereas the opposit e occurs when Mg-ATP is added to nucleotide-depleted F1. These spectro scopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes inv olving the epsilon-subunit.