Plants contain at least three kinds of hemoglobins. Those plants that carry out symbiotic nitrogen fixation use oxygen transport hemoglobins to deliver oxygen to the aerobic nitrogen fixing bacteria in their roots. The functions of other plant hemoglobins are not yet known with confidence, but are thought to also have roles in nitrogen metabolism. This dissertation examines plant hemoglobin structure and function in two distinct classes: oxygen transport hemoglobins and what we believe to be hemoglobins that function as dissimilatory nitrite reductases.

The capacity for oxygen transport arose twice independently in two distinct phylogenetic classes of plant hemoglobins. From "Class 2" hemoglobins arose the "leghemoglobins" common in many species of legumes including soybeans. From "Class 1" hemoglobins arose an individual oxygen transport hemoglobin in the species Parasponia andersonii (ParaHb). ParaHb and leghemoglobins have convergently evolved the clear physical properties supporting oxygen transport. Hemogobin from a closely related species, Trema tomentosa, is not an oxygen transporter, in spite of > 90% sequence identity to one another. The first part of this dissertation examines how such a small number of amino acid substitutions could result in the pronounced physical differences conferring a change in physiological function.

The second part of the dissertation presents evidence establishing dissimilatory nitrogen reduction as a physiological function for Class 1 plant hemoglobins. These proteins are able to efficiently reduce nitrite and hydroxylamine in vitro, in contrast to most other hemoglobins. It is shown that the unique structure of Class 1 plant hemoglobins facilitates catalytic reduction of nitrite and hydroxylamine by providing a ligand binding site and enhancing intermolecular electron transfer in support of multi-electron reduction reactions.