LIGAND-BINDING TO HEME-PROTEINS .3. FTIR STUDIES OF HIS-E7 AND VAL-E11 MUTANTS OF CARBONMONOXYMYOGLOBIN

Fourier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtaine d by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) sta te For most of the mutants, a single A substate band is observed, whic h points to the crucial role of the His-E7 residue in determining the A substate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclus ively connected to the presence of His-E7. In all but one mutant, mult iple stretch bands of the CO in the photodissociated state are observe d; these 8 substates are thought to arise from discrete positions and/ or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indi cate weak binding in the heme pocket. The observation of similar red s hifts in microperoxidase (MP-8), where there is no residue on the dist al side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to dete rmine the orientation of the bound ligand with respect to the heme nor mal by photolyzing small fractions of the sample with linearly polariz ed light at 540 nm. The resulting linear dichroism in the CO stretch s pectrum yielded angles alpha greater-than-or-equal-to 20-degrees betwe en the CO molecular axis and the heme normal for all of the mutants. W e conclude that the off-axis position of the CO ligand in the native s tructure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the dista l residue and the angle alpha of the CO ligand.