Crystal structure of a nonsymbiotic plant hemoglobin

Background: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant prot eins that is distinct genetically and structurally from leghemoglobins. The y are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O-2 conditions. Results: The first X-ray crystal structure of a member of this class of pro teins, riceHb1, has been determined to 2.4 Angstrom resolution using a comb ination of phasing techniques. The active site of ferric riceHb1 differs si gnificantly from those of traditional hemoglobins and myoglobins. The proxi mal and distal histidine sidechains coordinate directly to the heme iron, f orming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a nove l interface formed by close contacts between the G helix and the region bet ween the B and C helices of the partner subunit. Conclusions: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O-2 reversibly. However, the distal His73 is rapi dly displaced by ferrous ligands, and the overall O-2 affinity is ultra-hig h (K-D approximate to 1 nM). Our crystallographic model suggests that ligan d binding occurs by an upward and outward movement of the E helix, concomit ant dissociation of the distal histidine, possible repacking of the CD corn er and folding of the D helix. Although the functional relevance of quatern ary structure in nsHbs is unclear, the role of two conserved residues in st abilizing the dimer interface has been identified.