IMIDAZOLE IS A SENSITIVE PROBE OF STERIC HINDRANCE IN THE DISTAL POCKETS OF OXYGEN-BINDING HEME-PROTEINS

The FixL heme-based sensor, despite its low affinity for oxygen, is mu ch more reactive than myoglobin toward the large polar ligand imidazol e. To determine which features of a myoglobin heme pocket favor bindin g of imidazole, we have measured binding of this ligand to the FixL he me domain, elephant myoglobin, wild-type sperm whale myoglobin, and sp erm whale myoglobins having alanine, valine, threonine, glutamine, leu cine, phenylalanine, or tryptophan substitutions of the distal (E7) hi stidine residue. Except for histidine, the association rate constants dropped more than 3000-fold as the volume of the E7 side chain, at pos ition 64, was expanded from alanine (10(6) M-1 s(-1)) to phenylalanine (10(3) M-1 s(-1)). There was inhibition of imidazole binding due to d isplacement of coordinated water from H64 and H64Q sperm whale myoglob ins, where the E7 side chain hydrogen bends directly to the bound liga nd. The imidazole dissociation rate constants varied less dramatically and less consistently with any single factor, though they were measur ably decreased by hydrogen bonding to an E7 glutamine or histidine. On the whole, the results for the sperm whale myoglobin E7 substitutions show that the rate constants for imidazole binding are useful and sen sitive indicators of steric hindrance and polar interactions in the di stal pockets of myoglobins. The combined effects of the glutamine 64 a nd phenylalanine 29 in elephant myoglobin largely account for its incr eased imidazole association and dissociation rate constants, respectiv ely, compared to those of sperm whale myoglobin. An unhindered distal pocket not competent to stabilize positive poles is indicated by the l arge imidazole association (greater than or equal to 10(4) M-1 s(-1)) and dissociation (greater than or equal to 50 s(-1)) rate constants, p arameters that are characteristic of FixL.