Abstract 1970 Site-Directed Mutagenesis of Phytoglobins Leads to Increased Oxygen Dissociation Rates

All plants express proteins that are members of the globin family of proteins that fall into one of four evolutionary classes. The class 1 non-symbiotic hemoglobins, or phytoglobins, have a ligand binding site and oxygen binding kinetics that are not compatible with oxygen transport. These typically include a hexacoordinate heme iron, slow oxygen dissociation rates, and a high oxygen affinity. The symbiotic hemoglobins and class 2 non-symbiotic hemoglobins typically display features associated with oxygen transport. These features include: pentacoordinate heme irons, high concentrations, modest oxygen affinity and a relatively fast rate of oxygen dissociation. The class 3 plant hemoglobins more closely resemble truncated hemoglobins from bacteria and their structures and functions are still an active area of investigation. Prior studies have shown that the oxygen transport ability of hemoglobin in plants evolved in at least two convergent pathways, one from the class 2 globins, and a unique pathway from the phytoglobins. Phytoglobin from the plant Parasponia andersonii is a typical oxygen transport globin while the phytoglobin from Trema tomentosa has hexacoordinate heme iron and a low oxygen dissociation rate, both characteristics of a poor oxygen transporter. Interestingly, these two proteins differ in their primary structure by only 11 amino acids, providing a robust system for the studying structure and function relationships. The objective of our study is to determine which of the 11 amino acid differences between Trema tomentosa and Parasponia andersonii phytoglobins lead to changes in oxygen dissociation rate and physiological function. Based on previous work in our lab, two residues in critical areas of the globin fold were shown to increase oxygen dissociation rate. This study builds upon those results by using site-directed mutagenesis via PCR and the two residue mutant background to create a collection of triple mutants of Trema tomentosa. After purification by affinity chromatography, oxygen dissociation rates are measured by displacement with carbon monoxide in a stopped-flow reactor. The observed rates of these displacement reactions are used to calculate the rate of oxygen dissociation for the mutant and are then compared to oxygen dissociation rates of WT Trema tomentosa. Observed rates of oxygen dissociation in the triple mutants are faster than WT Trema phytoglobins which indicates a protein better suited for oxygen transport. These results begin to map the evolutionary pathway by which the oxygen transport function arose in phytoglobins and provide new insights into the relationship between primary structure and function in the globin family.