Role of Leu[superscript B 10] in cytoglobin and comparison of direct and indirect measurements of hexacoordinate hemoglobin ligand affinity

Hexacoordinate hemoglobins (HxHbs) have recently been discovered and have been found in nearly all living organisms. In humans, hxHbs are represented by neuroglobin (Ngb) and cytoglobin (Cgb). While Ngb is principally found in the brain, Cgb is ubiquitously expressed in all tissues. Ligand binding in hexacoordinate hemoglobins is complex due to the reversible coordination of the heme iron by the distal histidine. Hexacoordination regulates ligand binding and is regulated by specific residues in the heme pocket including the B10 position. Therefore, we investigated the influence of LeuB10 on hexacoordination and ligand binding in Cgb. Ligand binding was analyzed for recombinant proteins, Cgb B10A Cgb B10F. The results obtained showed that LeuB10 plays a crucial in hexacoordination. Interestingly, due to the mutants\u27 low CO affinity it was possible to measure the CO affinity equilibrium constant (Ka) by equilibrium titration similar to the Ka calculated from individual rate constants extracted from kinetics