The iron and subunit binding sites of hemerythrin. The role of histidine, tyrosine and tryptophan.
Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pk of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin. Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association. Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron.
D001665:Binding Sites; D002918:Chymotrypsin; D004355:Drug Stability; D006422:Hemerythrin; D006639:Histidine / Q000032:analysis*; D007501:Iron / Q000032:analysis*; D008667:Metalloproteins; D010084:Oxidation-Reduction; D010777:Photochemistry; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013774:Tetranitromethane
D006422:Hemerythrin; D008667:Metalloproteins; D006639:Histidine; D007501:Iron; D002918:Chymotrypsin; D013774:Tetranitromethane