| Summary: | The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the χ2obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes. The similarity between the values of the distribution centers of the long-lived components observed in the fluorescence decay of native myoglobins and those observed for apomyoglobins at acidic pH suggests that the long-lived component might arise from a conformational state (different from the native state) in which geometrical factors preclude energy transfer via Forster coupling from tryptophan to heme. This possibility has been further supported by the identification, in the steady-state emission spectrum of tuna myoglobin, of a component having an emission maximum similar to that observed at acidic pH, i.e., 335–337 nm. The difference between the tryptophanyl lifetime distributions of the two native holoproteins has been explained in terms of a higher degree of structural flexibility of tuna globin compared to that of sperm whale myoglobin. © 1989, American Chemical Society. All rights reserved.
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