RICE HEMOGLOBINS - GENE CLONING, ANALYSIS, AND O-2-BINDING KINETICS OF A RECOMBINANT PROTEIN SYNTHESIZED IN ESCHERICHIA-COLI

Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monoco ts and the function of Hbs in nonsymbiotic tissues, We report the clon ing and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least thre e copies of the hb gene were detected in rice DNA, and analysis of gen e expression shows that hb1 and hb2 are expressed in leaves but only h b1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escheri chia coli, and the recombinant Hb (rHb1) shows an unusually high affin ity for O-2 because of a very low dissociation constant. The absorbanc e spectra of the ferric and deoxyferrous rHb1 indicate that, in contra st to symbiotic Hbs, a distal ligand is coordinated to the ligand-bind ing site. Mutation of the distal His demonstrates that this residue co ordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O -2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that t his protein probably does not function to facilitate the diffusion of O-2.