Carbon monoxide binding to the ferrous chains of manganese,iron [Mn,Fe(II)] hybrid hemoglobins: pH dependence of the chain affinity constants associated with specific hemoglobin ligation pathways

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Abstract
In mixed-metal [Mn,Fe] hybrid [human] Hb, the 2 chains of a single type, .alpha. or .beta., are substituted with Mn protoporphyrin IX, which does not bind CO in either the Mn(II) or Mn(III) valency states. CO binding by the 2 Fe2+ subunits of a hybrid with Mn of either valency represents a simplified 2-step Hb ligation process in which ligands bind to a single-chain type. Considering the [Mn(II),Fe(II)] hybrids, which are deoxy T-state analogs, at pH 6.6 both types bind CO with low affinity (.alpha.-Fe, 0.38 mmHg; .beta.-Fe, 0.71 mmHg) and noncooperatively (Hill coefficient n =1). At elevated pH, both exhibit an increase in affinity (Bohr effect) and strong cooperativity, with the .alpha.-Fe hybrid having a higher degree of cooperativity (n .simeq. 1.6) than .beta.-Fe (.simeq. 1.3) at pH 9.0. The CO association constants for the Hb ligation routes in which the 1st 2 ligands bind to the same chain type are obtained from these measurements, and their pH dependence provides estimates of the proton release at each step. Through studies of CO on- and off-rates, the [Mn(III),Fe(II)] hybrids are used to obtain the pH dependence of the association constants for binding the 4th CO to the individual Hb chains. These results are used to parameterize an extended form of the 2-state, allosteric model for Hb cooperativity and provide the 1st direct determination of the pH dependence of the CO affinity constants for the individual chains in the T and R conformations, KT.alpha.(.beta.) and KR.alpha.(.beta.), the ratios of these constants, C.alpha. and C.beta., and the concentration ratio of the low- to high-affinity structural forms of unliganded Hb, L0. The .alpha. and .beta. chains show a similar T-state Bohr effect; at pH 7.1, ligation of either .alpha. or .beta. chains releases .apprx. 0.4 proton. In contrast, ligation of the chains within the R state releases at most .apprx. 0.2 proton at this pH. From pH 6.6-9.0, C.alpha. varies .apprx. 2-fold and C.beta. 4-fold. Even the extended Monod-Wyman-Changeaux model, which accounts for chain differences, incompletely describes Hb cooperativity, since multiple values of L0 are required to accommodate the data, and the suggestion of Weber regarding subunit interactions is supported.