Abstract 1524 Characterization of three bryophyte hemoglobin proteins

Agriculture is a rapidly developing field, and the continuing alteration of genes to create optimal yields has resulted in a need to better understand the physiological roles of plant proteins. An example of a plant protein in which the function remains unknown are non-symbiotic hemoglobins (nsHbs). In contrast to leghemoglobins, which are responsible for symbiotic oxygen transport in nitrogen fixing legumes, the function of most globin proteins in plant cells remains unknown. Hemoglobin (Hb) proteins in plants can be categorized by their coordination states and ability to bind different gasses that are biologically important in the plants such as oxygen, carbon monoxide, and nitric oxide. The globins of angiosperms and bacteria have been extensively studied in vitro, yet the information available on bryophyte globins, such as those from mosses, liverworts, and hornworts, remains scarce. The objective of this study is to determine if bryophyte hemoglobins display heme coordination and ligand binding characteristics consistent with or inconsistent with oxygen transport. UV-Vis spectroscopy and stopped flow kinetics were used to study heme coordination, auto-oxidation, and oxygen dissociation rates while flash photolysis was used to measure the rates of carbon monoxide association and oxygen association to the hemoglobin proteins. These measurements provide the means to compare moss (Physcomitrella), hornwort (Anthoceros) and liverwort (Marchantia) hemoglobin proteins to other non-symbiotic hemoglobins (nsHb) of later developed plants (rice) as well as mammalian globins (neuroglobin and myoglobin). Our results have shown an interesting combination of rate constants for binding biological gasses. When looking at the oxygen association rate of the bryophyte globins, it is comparable to that of the leghemoglobin family, at a rate between 150 μM-1 s-1 and 220 μM-1 s-1. Leghemoglobins are known oxygen transporters, leading to the conclusion that the hemoglobin protein in the bryophyte family could have some oxygen transport capacity. We also measured the carbon monoxide association rates, and these numbers are comparable to those of class 1 nsHbs, with most rates in this class falling between 0.5-5 μM-1 s-1. Class 1 nsHb globins are thought to have functions in nitric oxide scavenging in plants, and are not oxygen transporters. Leghemoglobins have average carbon monoxide association rates of 15 μM-1 s-1, much higher than our observed rates in the bryophyte globins. The bryophyte globins have been placed on phylogenetic trees as a common ancestor to the leghemoglobin and Class 1 nsHb proteins, and our data supports this placement. Results of our kinetic measurements suggest that the bryophyte family of globins holds a position at the point of divergence between oxygen transport and non-oxygen transport in the evolution of plant hemoglobin proteins. Thank you to Drake STAR Fund for travel funding.