INVESTIGATION OF HEME-PROTEIN INTERACTIONS IN TRUNCATED HEMOGLOBINS

Fe-protoporphyrin IX, otherwise known as heme b, is found in all hemoglobins. The heme iron is coordinated by four nitrogen atoms from the tetrapyrrole ring of protoporphyrin IX. In hemoglobin the heme is bound to the protein via a proximal histidine residue, resulting in a 5-coordinate heme. Many hemoglobins also have a sixth distal ligand in the form of an exogenous ligand, such as water or oxygen, or, in some cases, a protein residue. Despite having highly conserved tertiary structure and the same cofactor, hemoglobins display a variety of functions, such as oxygen transport, oxygen sensing, and nitric oxide scavenging. Hemoglobins and heme proteins in general are capable of controlling the chemistry performed by the heme, and therefore the function of the protein, via two primary methods: posttranslational modification (PTM) and axial ligation. In two cyanobacterial truncated hemoglobins (trHbs, from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, collectively named GlbN), when the heme contains a ferrous iron and is not carrying oxygen or carbon monoxide, a covalent bond forms between the heme-2 vinyl and a nearby histidine residue. The PTM in GlbN has been extensively studied and these studies show it is not dependent on the distal endogenous ligand and the local environment around the reactive histidine. To confirm that the main determinant of PTM is the relative positions of the reactants I engineered the PTM in the heme domain of Chlamydomonas eugametos LI637 protein (CtrHb). CtrHb is structurally similar to GlbN, sharing an average ~46% identity with them. The differences between CtrHb and GlbN are distributed throughout the structures and include some the heme pocket. Of the 21 residues that are in contact with the heme, 10 are different between the two proteins. This difference provides an altered chemical environment in which to test the reaction that generates the PTM. Enhanced chemiluminescence detection of the heme in reacted CtrHb variants shows that it is possible to engineer the PTM in this protein. This suggests that the heme pocket of GlbN does not greatly contribute to the PTM reaction and that the PTM could be engineered in other proteins. The nature of the distal heme ligand is variable and can affect the rate exogenous ligand binding, the electron transfer rate, and the redox potential of heme proteins. Owing to the importance of the distal ligand, special attention was placed on identifying the distal ligand during the optical and crystallographic characterization of the eukaryotic trHb from Chlamydomonas reinhardtii, THB1, in addition to investigating the function of this protein. In this work I expanded upon our knowledge of these two heme proteins, CtrHb and THB1, in order to gain a better understanding of how proteins control heme reactivity