An EPR study of manganese(II) binding to 5′-ATP, hemoglobin, and hemocyanin

Abstract

Several divalent metal ions affect the oxygen affinity of hemoglobin and hemocyanin. It is important, therefore, to understand the nature of metal-ion binding to these proteins. By comparing the EPR spectra of Mn(II), 0.001 M, in the absence and presence of carboxyhemoglobin or Limulus oxyhemocyanin (pH 7.3, Trizma buffer), the number of Mn binding sites, n, and the binding constant, K, can be determined. For carboxyhemoglobin, HbCO, we find 0.5 Mn binding sites per heme, K = 450 M −1. Each hemoglobin tetramer therefore binds two manganous ions suggesting that Mn(II), like Cu(II), may bind preferentially to one of the two types of subunits in hemoglobin. For hemocyanin, HcO 2, we find n = 5.8, K = 1.55 × 103 M −1. Each oxyhemocyanin therefore binds approximately six manganous ions, and the binding constant is three times larger than that for HbCO. We have also carried out similar experiments on 5′-ATP, and on solutions of HbCO and ATP containing MgCl 2 or ZnCl 2. Zn(II) effectively competes with Mn(II) in binding hemoglobin and ATP, whereas Mg(II) does not, in accord with expectations from data on oxygen affinity of hemoglobin.