Molecular Dynamics Reveals Unique Distal Histidine Positions and Interfacial Transitions of Fish and Mammalian Deoxyhemoglobin at Post-mortem pH.

Molecular dynamics simulations were utilized to evaluate the mechanisms that cause trout IV Hb to have enhanced oxidative capacity compared to bovine Hb. The focal points of analysis were amino acid differences in the heme pocket and at the α1-β2 interface and the effect of varying distal histidine protonation state. Unprotonated HisE7 that swung up came back down only in bovine Hb, attributable to weaker π-π stacking interactions for PheCE4 (bovine) compared to TrpCE4 (trout IV). Protonation of HisE7 causes this residue to stay pointed down toward the heme only in bovine DHb. This finding was complimented by experimental findings indicating elevated nitrite reductase activity of bovine DHb at low pH, highlighting the importance of the HisE7 orientation for catalysis. Computational analysis indicated key interfacial differences that may promote tetramer disassembly of fish DHb at a low pH. Size exclusion chromatography data corroborated this finding, showing a differential size distribution between fish and mammalian DHb at pH 5.7. Overall, these studies suggest that the elevated pro-oxidative capacity of trout IV Hb is related to an increased tendency for subunit formation and a greater propensity for HisE7 to be in the "up" position providing a channel for oxidants to enter the heme pocket.