Quaternary structure of rice nonsymbiotic hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.