Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography

The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F_o - F _c and 2F_o-F_c neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α945; or Β chains, α945;His20, α945;His50, α945;His58, α945;His89, ΒHis63, ΒHis143 and ΒHis146, have different protonation states. The proton-ation of distal His residues in the α945;1Β1 heterodimer and the protonation of α945;His103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their α916;pK a between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.