Interaction between bound cupric ion and spin-labeled cysteine β-93 in human and horse hemoglobins

Abstract

The location of the various copper binding sites for horse and human hemoglobin was probed using spin labels attached to the β-93 cysteine residue. Dipole-dipole interactions between the spin label and bound copper produce a decrease in the amplitude of the spin label spectrum which was used to estimate the Cu(II) spin label distance. By comparing the results with horse and human hemoglobin at 298 and 77 K four different Cu(II) binding sites were identified. The low affinity horse hemoglobin site with the sulfhydryl blocked (site 1) was found to be located 10–13 Å from the sulfhydryl spin label on the surface of the molecule. Only with a free sulfhydryl is the site (site 2) in the pocket between the F and H helices closer to the SH-group and the iron populated. It is site 2 which is responsible for the oxidation. In frozen solutions a Cu-nitroxide distance of about 17 Å was determined with human hemoglobin. This distance is consistent with the previously postulated location of the “high affinity” human hemoglobin site near the amino terminus of the β-chain. At 298 K a much shorter Cu-nitroxide distance of about 7 Å was calculated for human hemoglobin. This shorter distance at higher temperature also correlated with a slightly smaller value of g₁₁ and A₁₁ for the Cu(II) ESR spectrum. It is postulated that in solution cross-linking between nitrogenous ligands in the region of the amino terminus of one β-chain and the carboxyl terminus of the other β-chain can explain this shorter distance. This crosslink could involve histidine β-143, which is one of the ligands thought to be also involved in site 1. Binding to the “high-affinity” site in solution thus stabilizes the “low affinity” site 2 relative to site 1 explaining the reported interaction between the “high-affinity” and “low affinity” sites.