Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.

摘要

Two rice (Oryza sativa) haemoglobin (Hbs) genes, hb1 and hb2, that code for plant Hbs, were cloned and analysed. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression showed that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) showed an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicated that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal Hisdemonstrates that this residue coordinates the haeme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2. The sequences for the hb1 and hb2 genes and Hb1 and Hb2 cDNA have been deposited in the GenBank database under accession numbers U76028, U76029, U76030 and U76031, respectively.