The contribution of histidine (HC3) (146β) to the R state Bohr effect of human hemoglobin

The role of histidine (HC3) 146 β in the previously established pH-dependent properties of the R state of human hemoglobin has been investigated. The rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l₄ and l'₄, have been determined as a function of pH for hemoglobin A and des-(His 146 β) hemoglobin A. From these kinetic parameters, the value of L₄, the affinity constant for the 4th carbon monoxide molecule, has been calculated according to the equation L₄ = l'₄/l₄. In addition, the effect of removal of histidine 146 β or its replacement by arginine (hemoglobin Cochin-Port Royal) on k₄, the rate of oxygen dissociation from fully liganded hemoglobin, has been determined as a function of pH. Removal of histidine 146 β reduces the pH dependence of L₄ by 47%. At the same time, it produces a similar, 45%, reduction in the pH dependence of k₄. Replacement of histidine 146 β by arginine reduces the pH dependence of k₄ by 23% and that of l'₄ by about 30%. These chemical modifications cause reductions in the R state Bohr effect which are remarkably similar in magnitude to the reduction which they produce in the overall Bohr effect. These results indicate that histidine 146 β controls a major fraction of the R state Bohr effect as well as being a major participant in the overall and T state Bohr effects.