HEME-CO BINDING TO TRYPTOPHAN-CONTAINING CALMODULIN MUTANTS

The binding of heme-CO to genetically engineered calmodulin containing a single tryptophan residue has been studied. A tryptophan residue wa s integrated at one of five positions: 26 or 62 of the N-terminal, 81 in the central helix, or 99 or 135 of the C-terminal. As for the wild type, the mutant calmodulins bind four molecules of heme-CO with an av erage affinity of 1 mu M. (i) Homotropic effect, The quenching of the tryptophan fluorescence by energy transfer to the hemes indicates that there is no preference between the N- or C-terminal pockets for heme binding. The quenching is less than expected for a binomial distributi on of four sites. This could indicate a lower energy transfer rate due to a specific orientation factor. The weak quenching as a function of the number of hemes bound may also reveal a cooperativity in the heme binding; the data can be simulated assuming two pairs of sites, where each pocket shows a cooperative binding for two hemes. (ii) Heterotro pic effect. As observed for the wild type, addition of melittin does n ot displace the hemes from the mutant calmodulins; the affinity of hem e-CO for the calmodulin melittin complex is higher than that for calmo dulin alone. The affinity of heme-CO for native calmodulin is also hig her in the presence of trifluoperazine.