TIME-RESOLVED EMISSIONS IN THE PICOSECOND RANGE OF SINGLE TRYPTOPHAN RECOMBINANT MYOGLOBINS REVEAL THE PRESENCE OF LONG-RANGE HEME PROTEIN INTERACTIONS

We have analyzed the time resolved fluorescence emission in the subnan osecond range of recombinant wild-type SW myoglobin and its single TRP mutants W7F and W14F. These recombinants carry a methionine at the N1 terminal end. The emission of Trp-7 in the met form of W14F showed re sidual lifetime components much shorter than those estimated after exc itation energy transfer to the heme. We propose that in this recombina nt the N1 methionine is close to Trp-7, thereby producing an extra que nching due to either collisions or electron transfer with its sulfur. When the measurements were repeated on its GO-form, the extra quenchin g of Trp-7 was much decreased, indicating a heme linked conformational change involving the amino terminal end of the protein. This hypothes is is supported by ligand linked conformational changes in myoglobin, reported by Ansari et al. and by Giardina et al, At neutral pH the lif etimes of W7F were consistent with estimations based on the atomic coo rdinates of SW myoglobin. Those of the wild-type were exactly the comb ination of the lifetimes of the two mutants. This suggest that the mut ations did not affect the overall structure of the protein. However, i n the ferric form, substitution of Trp-14 in W14F resulted in low stab ility at acid pH, as evident from lifetimes modifications at pH 4.8, w hile no modifications were produced by titrations of W7F to pH 4.5. Th is suggests a role of Trp-14 in the structural stability of myoglobin. (C) 1998 Elsevier Science B.V. All rights reserved.