We have identified the steady-state and time-resolved fluorescence of the three tryptophan residues (Trp-86, Trp-130, and Trp-140) of the pore-forming domain of colicin A using site-directed mutagenesis in order to construct two- and one-tryptophan-containing mutant proteins. Fluorescence lifetimes were measured via multifrequency phase fluorometry. The fluorescence of the pore-forming domain of colicin A is dominated by Trp-140 which contributes almost 53% to the fluorescence intensity. Mutation of Trp-140 results in a decrease in fluorescence quantum yield and average lifetime. Colicin A wild-type and all mutant proteins display multiple lifetimes which belong to three different lifetime classes: 0.38-0.57 ns for tau(1), 1.6-1.87 ns for tau(2), and 3.6-4.41 ns for tau(3) at pH 5. At pH 7, the three classes are 0.64-0.89 ns for tau(1), 2.01-2.19 ns for tau(2), and 4.23-4.94 ns for tau(3). This pH effect influences all the lifetimes and must be attributed to a general conformational change. In wild-type colicin A, tau(3) originates mainly from Trp-140 while Trp-86 and Trp-130 both provide a major contribution to tau(2). The pH dependence of the fluorescence intensity gives rise to a pK(a) of 5.2. The different lifetime components of two of the three single-tryptophan-containing mutants show different quenching properties toward acrylamide, indicating that each lifetime is coupled to a different microenvironment. The linear combination of the lifetimes of the single tryptophans into pairs simulates very well the behavior of the two-tryptophan-containing mutants except for one, the mutant containing Trp-86 and Trp-130. The lifetimes of the wildtype protein can only be obtained by the linear combination of the lifetimes from the mutant containing the tryptophan pair Trp-86/Trp-130 and the mutant containing Trp-140. Mutual energy transfer between Trp-86 and Trp-130 is assumed to be the explanation of this deviation since the mutant proteins display no structural or dynamic aberrances. The calculated energy transfer efficiency amounts to 65% for energy transfer from Trp-86 to Trp-130 and 21% for the reverse transfer and is in agreement with our measurements.