Bile-pigment formation from different leghaemoglobins. Methine-bridge specificity of coupled oxidation

Abstract

The coupled oxidation of leghaemoglobins with O(2) and ascorbate yielded oxyleghaemoglobin in the first reaction step, and the second step was the degradation of haem characterized by an A(675) increase. Leghaemoglobins were degraded to biliverdin isomers specifically, depending on the structure of the protein. The main leghaemoglobin components of Glycine (soya bean) and Phaseolus (kidney bean) were degraded to biliverdin mixtures containing about 50% of the beta-form, about 30% of the alpha-form and about 20% of the delta-isomer, whereas the leghaemoglobin I components of Vicia (broad bean) and Pisum (pea) were degraded almost exclusively to the beta-isomer, with traces of the alpha-isomer. The amino acid sequences of Glycine and Phaseolus leghaemoglobins resemble each other, as do those of Vicia and Pisum. The site specificity of bile-pigment formation from leghaemoglobins can be tentatively explained by specific differences in the amino acid sequences at those regions of the polypeptide chain that are in the vicinity of the appropriate methine bridges. The ligand-binding site in different leghaemoglobins may be outlined on the basis of the present results, supposing that the haem is degraded when a reduction product of haem-bound O(2) reacts with a methine bridge of the haem, and that the bridge specificity is regulated by hindering amino acid residues that determine the location of the bound O(2). The residue phenylalanine-CD1 appears to be further away from the haem plane or in a markedly more flexible position in leghaemoglobins than in mammalian globins. The haem-bound oxygen atom B, in Fe-O(A)-O(B), seems to be free to rotate in all directions except that of the gamma-bridge in Glycine and Phaseolus leghaemoglobins, but its position in Vicia and Pisum leghaemoglobin I might be restricted to the direction of the beta-methine bridge.