Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts

• pH-regulated iron coordination state of hemoglobins monitored by Soret peak shifts.
• 5- and 6-coordinate globins display tri- and bi-phasic pH profiles, respectively.
• The pH profiles are diagnostic plots for 5- and 6-coordinate ferric hemoglobins.
• The pH dependence implies heme coordination regulation with functional relevance.
• In long-term, the pH profiles may have value in diagnosis of hemoglobin pathology.

Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.