Quaternary structure has little influence on spin states in mixed-spin human methemoglobins

Abstract

A key feature of the Perutz stereochemical model for cooperativity in Hb is a strong coupling between quaternary structure and the spin state of the heme Fe. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin .dblarw. low-spin equilibrium. To test this hypothesis, the changes were measured in spin equilibria upon conversion of 6 mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20.degree. C in 20 mM maleate buffer, ph 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz sterochemical model. These data do not support a view that the low affinity of the T state is due to restraints acting through the Fe proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.