Human neuroglobin and H64A distal variant: how mutation and pH affect the heme pocket

Neuroglobins (Ngbs) are low spin heme proteins, with His64 and His96 residues bound to the distal and proximal positions of the heme iron, respectively. His64 must be displaced to allow the ligation of exogenous molecules to the heme, thus regulating ligand binding. In fact, in the absence of the distal His, at physiological pH, the protein becomes high spin in both the ferric and ferrous states. However, the variant undergoes a pH-dependent coordination/spin state transition to a hexacoordinated low-spin species at alkaline pH, featuring Lys67 as the distal endogenous ligand. Here, we have investigated the effect of pH on the wild-type (WT) protein and the distal H64A variant of human Ngb in solution by a combination of UV–Vis electronic absorption, Magnetic Circular Dichroism and resonant Raman spectroscopies. In the WT protein, only a vinyl substituent conformational change is observed with increasing pH, due to the strong stability of the bis-histidyl heme coordination. Conversely, in the H64A variant alkaline pH induces a major structural rearrangement of the active site with the distal coordination of Lys67 to the heme, a change in the strength of the hydrogen bonds of the propionate groups and a conformational rearrangement of one vinyl substituent. We propose that these findings are due to the modifications in the E-helix as a consequence of Lys67 deprotonation in both the WT and the variant. These results provide further details at the molecular level on the fundamental role of distal residues for heme binding within the active site and protein stability.