Metal ion coordination in ‘R’ and ‘T’ state hybrid hemoglobins as revealed by optical, EPR and sulphhydryl reactivity studies

The sulphhydryl environment in various mixed-metal hybrid hemoglobins, viz. α2(Cu)β2(FeCO), α2(FeCO)-β2(Cu), α2(Cu)-β2(Ni), α2(Ni)-β2(Cu), was studied by reacting them with the sulphhydryl reagent, 4,4′-dithiodipyridine (4-PDS). The reactivity was compared with that of HbCO, NiHb and CuHb. It is found that there exists a correlation between conformational change and metal ion environment, not only at the extreme R and T states but also the intermediate conformations. EPR examinations of these hybrids show that both in R state-[Cu(II)-Fe(II)] and T state-[Cu(II)-Ni(II)] hybrids at neutral pH and in the absence of IHP, CuPPIX, irrespective of the subunit in which it is present, has a mixed-metal ion environment: Species 1, a five-coordinated Cu complex with strong proximal histidine bond and species 2, a four-coordinated complex without any covalent linkage with Nε F8-histidine.