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The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe–F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe–F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe–F) wavenumbers, an empirical correlation has been found. The …