Primitive haemoglobin

Summary The sequences ofPetromyzon andAplysia globins are compared with the postulated vertebrate and mollusc-vertebrate ancestors to see if differences exist in the rates of evolution of different types of residue positions. Between the mollusc-vertebrate ancestor andAplysia globin there is no very striking pattern of changes except that the interior positions are relatively conserved. In the evolution ofPetromyzon haemoglobin, the _{1 2} contact area is relatively conserved. The homopolymeric binding of lamprey Hb seems to be a primitive function.     

Effect of ionizing radiation on haemoglobin: the oxy-derivative of haemoglobin Iwate

It is well established that exposure of oxyhaemoglobin to ionizing radiation results in remarkably selective electron addition to the (FeO2) unit, giving a novel species, (FeO2)-, in which the extra electron is largely localized on iron and dioxygen. This work has now been extended to haemoglobin (Hb.) Iwate. The haemoglobin M. Iwate used is a mutant haemoglobin having only Fe(III) alpha-chains by oxy beta-chains (alpha 2 Met beta 2 oxy). The haem iron atoms in the alpha-chains are coordinated in the fifth site by a proximal tyrosine in place of histidine. This unit is a high-spin Fe(III) with axial symmetry and prominent electron spin resonance (ESR) features in the g = 6 and g = 2 regions. On exposure to 60Co gamma-rays at 77 K, efficient electron addition occurred at both types of iron centre, giving Fe(II) and (FeO2)- units. The former was monitored by the decrease of the g = 6 feature for Fe(III) and the latter by the growth of g-features at 2.254 (gx), 2.149 (gy) and 1.967 (gz). These values are close to those for the FeO2- centre formed in the beta-chains of normal oxyhaemoglobin. On annealing above 77 K, two changes occurred: first there was a small but clear increase in gx and gy, followed by a marked reduction in gx and gy giving g-values close to those for the centre formed directly in the alpha-chains of the normal protein. Finally, this intermediate species gave a centre having gx = 2.310, gy = 2.180 and gz = 1.935. These values are typical of low-spin Fe(III) haemoglobin and are assigned to the protonated complex, Fe(III)O2H. Ultimately at ca. room temperature, this was converted into the high-spin, met-form, with a gain in the g = 6 feature. These results established that the beta-chain centre in Hb. Iwate behave in the same way as isolated beta-chains. They also confirm that electron addition to the oxy-units is facile, even in the presence of Fe(III) units in each tetramer. The results also confirm that electron capture to give (FeO2)- units is not followed by internal electron-transfer to give Fe(II) from the Fe(III) centres in the alpha-chains.     

Molecular evolution of plant haemoglobin: two haemoglobin genes in Nymphaeaceae Euryale ferox

We isolated and sequenced two haemoglobin genes from the early-branching angiosperm Euryale ferox (Nymphaeaceae). The two genes belong to the two known classes of plant haemoglobin. Their existence in Nymphaeaceae supports the theory that class 1 haemoglobin was ancestrally present in all angiosperms, and is evidence for class 2 haemoglobin being widely distributed. These sequences allowed us to unambiguously root the angiosperm haemoglobin phylogeny, and to corroborate the hypothesis that the class 1/class 2 duplication event occurred before the divergence between monocots and eudicots. We addressed the molecular evolution of plant haemoglobin by comparing the synonymous and nonsynonymous substitution rates in various groups of genes. Class 2 haemoglobin genes of legumes (functionally involved in a symbiosis with nitrogen-fixing bacteria) show a higher nonsynonymous substitution rate than class 1 (nonsymbiotic) haemoglobin genes. This suggests that a change in the selective forces applying to plant haemoglobins has occurred during the evolutionary history of this gene family, potentially in relation with the evolution of symbiosis.     

Hydroxylase activity of immobilized haemoglobin

Until now, hydroxylation of substrates for practical applications has been mostly carried out by chemical and microbial processes. The hydroxylase activity of haemoglobin could be of great help for this purpose. Hydroxylation of aniline by haemoglobin immobilized as cross-linked soluble polymers and insoluble particles was studied. Activity yields after immobilization as well as kinetic constants were estimated. Hydroxylase activities similar to those of liver microsomal cytochrome P-450 activities were obtained.