CHARACTERIZATION OF RECOMBINANT SOYBEAN LEGHEMOGLOBIN A AND APOLAR DISTAL HISTIDINE MUTANTS

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodu le cDNA library and expressed in Escherichia coli. The crystal structu re of the ferric acetate complex of recombinant wild-type Lba was dete rmined at a resolution of 2.2 Angstrom. Rate constants for O-2, CO and NO binding to recombinant Lba are identical with those of native soyb ean Lba. Rate constants for hemin dissociation and auto-oxidation of w ild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale m yoglobin due both to a fourfold higher rate of auto-oxidation and to a similar to 600-fold lower affinity for hemin. The role of His61(E7) i n regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in t he equilibrium constant for O-2 binding to soybean Lba, whereas the sa me mutations in sperm whale myoglobin cause 50 to 100-fold decreases i n K-O2. These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O-2 binding to the soybea n protein. The His(E7) to Phe mutation does cause a significant decrea se in K-O2 for Lba, apparently due to steric hindrance of the bound li gand. The rate constants for O-2 dissociation from wild-type and nativ e Lba decrease significantly with decreasing pH. In contrast, the O-2 dissociation rate constants for mutants with apolar E7 residues are in dependent of pH, suggesting that hydrogen bonding to the distal histid ine residue in the native protein is enhanced under add conditions. Al l of these results support the hypothesis that the high affinity of Lb a for oxygen and other ligands is determined primarily by enhanced acc essibility and reactivity of the heme group. (C) 1997 Academic Press L imited.