Hemoglobin has been one of the most studied proteins due to its vital function of blood oxygen transport as well as its exemplary quaternary structure and allosteric functions (1). There are multiple different conformations and forms of hemoglobin given its tetrameric structure and ability to bind multiple types of molecules. In particular, the human deoxyhemoglobin form (PBD ID: 1bz0) is of interest. This protein’s primary function is to bind oxygen molecules found in the lungs, and to transport that oxygen to tissues and organs throughout the body (2). Hemoglobin proteins are often compared to myoglobin, a related oxygen storage protein found in high amounts in skeletal muscle. The particular deoxyhemoglobin studied was taken from humans, increasing its biological significance for the understanding of human physiology, as well as in a medical or healthcare context. 

Multiple databases were used in order to investigate the structure, primary and secondary, and general physical characteristics of deoxyhemoglobin. In order to find relevant comparison proteins, PSI-BLAST and the Dali server were used. PSI-BLAST finds proteins with similar primary sequence to a protein query. An E value of each resulting protein quantifies the number of gaps representing mismatched amino acids, giving a lower E value (less than 0.05 is siginifcant) for most similar proteins. The Dali server is more structurally oriented in that it compares intramolecular distances of tertiary structures of proteins to a protein database. A Z-score above 2 indicates that proteins have significantly similar folds (3). 

In its physiological form, deoxyhemoglobin is a globular protein found in the red blood cells of the blood stream (4). Deoxyhemoglobin is the form of hemoglobin that is missing the oxygen bound to the heme ligand groups, also called the T (tense) state (1). Deoxyhemoglobin is a tetrameric structure containing 2 alpha subunits and 2 beta subunits interacting in a symmetric plane. The molecular weight of deoxyhemoglobin is 46084 Da, and the isoelectronic point is 8.48 (5). Each alpha subunit contains 141 residues, and each beta subunit has 146 residues (6).  The interactions of the subunits are usually between alpha and beta chains, with very few alpha-alpha or beta-beta associations. The four subunits form a tetrahedral arrangement, giving a spherical protein with a large water cavity relative to that of the oxygen-bound hemoglobin (1). Eight alpha-helical sections lettered A-H compose each alpha and beta subunit, and each subunit of the tetrameric deoxyhemoglobin is characterized by specific helix section interactions. Especially important contacts occur between helices B, G, and H, which do not change when the heme groups bind oxygen (1). However, when deoxyhemoglobin binds oxygen the C and G helix interface is altered and experiences a conformational shift (1). 

The secondary structures present in deoxyhemoglobin are alpha helices, 3/10 helices and random coils. As mentioned before, the eight alpha helices interact with one another determining the dimer associations of the protein. Random coils do not account for much of the secondary structure composition of deoxyhemoglobin. One mutant of deoxyhemoglobin is hemoglobin Catonsville, in which an inserted glutamate residue affects the turns of the 3/10 helices (6). These helices may adopt a shifted conformation as a consequence of the inserted residue between Pro-37 and Thr-38. The subunit interactions of tetrameric deoxyhemoglobin with each other and with each of the four heme groups are driven by critical residues and salt bridges (1). In the alpha chains N-terminus and C-terminus interactions of Arg-141, Val-1, Asp-126, Lys-127, Val-34, Val-93, and Tyr-140 form non-covalent electrostatic interactions. On the beta chains, interactions between Tyr-145, His-146, Asp-94, and Lys-40 serve the same function. Hydrogen bonds between beta chain Tyr-145 and Val-98 are one of the structural characteristics disrupted by the binding of oxygen. His-87 binds the heme group of the alpha subunits whileHis-92binds the heme group of the beta subunits (7). 

The only ligand present in deoxyhemoglobin is the heme group. The whole protein contains four heme groups in total, with one on each subunit. The heme group provides deoxyhemoglobin’s main physiological function in binding oxygen for transport throughout the body. The heme group is an iron-porphyrin IX complex containing a ferrous iron molecule that interacts with the nitrogen atoms of imidazole atoms of histidine residues (4). The ferrous iron binds a gas on the other side of the bond plane- usually oxygen but carbon dioxide, carbon monoxide and nitrogen monoxide can also be transported. The binding of oxygen gas to the ferrous iron of the heme group converts deoxyhemoglobin to hemoglobin (PDB ID: 1GZX), and induces a conformation change. The Fe2+-His-N bond is tilted due to steric strain and pulls the histidine residue as it readjusts closer to the porphyrin plane, causing a shift of helix F which causes the subunit interfaces to adjust as well (1). This shift affects the entire subunit, breaking some interchain salt bridge linkages between residues.

          Aside from providing 70% of the iron in human blood, deoxyhemoglobin gives insight into mechanisms of higher protein structure and the structural adaptations that arise from ligands that bind several substances.  Therefore, the study of deoxyhemoglobin can be deemed extremely biologically significant in that it gives insight to a protein vital to human blood and valuable representation of themes of protein mechanisms and structures in general due to its quaternary structure and binding activity (2). 

The PSI-BLAST and Dali server results directed the investigation of a comparison protein by finding proteins with similar primary sequences and tertiary structures. The comparison protein chosen, aquomet porcine hemoglobin (PBD ID: 2pgh), had an E value of 2x10-79 in the PSI-BLAST and a Z score of 26.6 on the Dali server (3). Both of these values deem aquomet porcine hemoglobin a structurally significant similar protein to deoxyhemoglobin. Aquomet porcine hemoglobin provides an alternate example of a hemoglobin molecule from a different species, Sus scrofa. Like deoxyhemoglobin, it has 4 heme groups but since it is in the R (relaxed) state, the iron of the heme groups has bound oxygen. The structure of this porcine hemoglobin shows an 85% sequence similarity to human hemoglobin (9). The dissimilar portion of the sequence structure arises from the inhibition of the alkaline Bohr effect and a reduction of oxygen-linked chloride binding (9). In human hemoglobin, the Bohr effect is a release of protons from binding oxygen as residues of salt bridges alter their pK's when their linkages are broken. The comparison protein has biological significance due to the increased interest in lower mammal hemoglobin structures to be used in the possible development of blood substitute substances for humans. In addition, the structural similarities between the two species of hemoglobin/deoxyhemoglobin give insight to the results of attempted hybridized expression of human hemoglobin in the red blood cells of transgenic pigs.  In these attempts, the resulting porcine erythrocytes exhibit qualities of both human and porcine hemoglobin. 

The study of deoxyhemoglobin is biologically significant due to its abundance across multiple species and functional importance to oxygen transport throughout the blood stream. Comparing hemoglobin derivative structures across species provides support of the evolutionary significance of the protein in a biological context as well. Hemoglobin's structure and allosteric behavior makes it a good example of classic protein themes seen across mulitple types of molecules. The quaternary symmetrical tetramer serves as a model of typical quaternary association of proteins. Subunit interactions with one another drive them to associate and form the tetramer. Hemoglobin's conformational shifts after binding a substrate such as oxygen gives examples of the mobility and versatility of protein structures interacting with bound molecules. The presence of hemoglobin’s tetrameric quaternary structure across multiple species proves that it is a functionally significant example of protein folding and association in higher order structures.