No abstract

The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

No abstract

The low-molecular-mass fraction of the soybean nodule cytosol contains Fe capable of catalyzing free radical production through Fenton chemistry. A large portion of the pool of catalytic Fe, measured as bleomycin-detectable Fe, was characterized as complexes of Fe with phenolic compounds of three classes: phenolic acids, cinnamic acids, and flavonoids. Many of these compounds, along with other phenolics present in legume tissues, were used for a systematic structure-activity relationship study. All phenolics tested were able to chelate Fe, as judged from their inhibitory effect on site-specific deoxyribose degradation (minus EDTA assay). However, only those having catechol, pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe 3+ at pH 5.5.The same phenolics promoted oxidative damage to DNA (bleomycin assay) and to deoxyribose (plus EDTA assay), but inhibited linolenic acid peroxidation by chelating and reducing Fe 3+ and by neutralizing lipid radicals. Also, phenolics having a pyrogallol nucleus attenuated the free radical-mediated inactivation of glutamine synthetase, which was used as a model system, by chelating Fe 2+ . It is reasoned that under the microaerobic (10-20 nM O 2 ) and acidic (pH 5.5-6.4) conditions prevailing in nodules, phenolics are likely to act primarily as antioxidants, decreasing oxidative damage to biomolecules.

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-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 myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.