The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study

The paramagnetic species in human metHb and horse metmyoglobin (metMb) have been studied at low temperature using EPR spectroscopy. The high-spin (HS) haem signal in aquometMb has a greater rhombic distortion than the HS metH b signal. Nevertheless, the individual line width (g = 6) is smaller in met Mb than in metHb, consistent with non-identical signals from the alpha and beta Hb subunits. Three low-spin (LS) haem forms are present in metHb, whil e metMb has only two. The major LS form in both proteins is the alkaline sp ecies (with OH- at the sixth co-ordination position). The minor LS forms ar e assigned to different histidine hemichromes in equilibrium with the norma l HS species at low temperature. LS forms disappear when the haem is bound by a ligand, such as fluoride, which ensures 100% occupancy of the HS state both at room temperature and at 25 K. The small differences in effective g -factors of the histidine hemichromes are interpreted in terms of different distances between the distal histidine and haem iron. The pH dependence of the inter-conversion of the different paramagnetic species is consistent w ith a model whereby protonation of a residue with a pK of 5.69 (metHb) or 6 .12 (metMb), affects ligand binding and transformation from the HS to the L S form. Chemical and spectroscopic considerations suggest that the residue is unlikely to be the proximal or distal histidine. We therefore propose a model where protonation of this distant amino acid causes a conformational change at the iron site. Identical effects are seen in frozen human blood, suggesting that this effect may have physiological significance.