Time resolved emissions of single tryptophan myoglobins reveal long range heme protein interactions

We have analyzed the time resolved fluorescence emission in the subnanosecond range of recombinant wild-type SW myoglobin and its single TRP mutants W7F and W14F . These recombinants carry a methionine at the Nl terminal end. The emission of Trp7 in the met form of W14F showed residual lifetime components much shorter than those estimated after excitation energy transfer to the heme. We propose that in this recombinant a loop brings the Nl methionine close to Trp7, thereby producing an extra quenching due to either collisions or electron transfer with the sulfur of the N l-methionine. When the measurements were repeated on its CO-form, the extra quenching of Trp7 was much decreased, indicating a heme linked conformational change involving the amino end of the protein- This hypothesis is supported by ligand linked conformational changes in myoglobin, reported by Ansari et al (Biochemistry 35:5128, 1994) and by Giardina et al (J.Biol.Chem. 29:16999, 1996). At neutral pH the lifetimes of W7F were consistent with estimations based on the atomic coordinates of SW myoglobin. Those of the wild-type were exactly the combination of the lifetimes of the two mutants. This suggest that the mutations did not affect the structure of the protein. However, substitution of Trp-14 in W14F resulted in low stability at acid pH, as evident from lifetimes modifications at pH 4.8, while no modifications were produced by titrations of W7F to pH 4.5. This suggests a roîe of Trp-14 in the structural stability of myoglobin.