MERCURY BINDING TO HEMERYTHRIN - COORDINATION OF MERCURY AND ITS EFFECTS ON SUBUNIT INTERACTIONS

The reaction of hemerythrin, an oxygen-carrying protein in marine invertebrates, with K2HgI4 was investigated to determine the mode of mercurial binding to the protein in the heavy atom derivative used for solving the crystal structure of Themiste dyscritum hemerythrin. Spectrophotometric titrations established that the T. dyscritum protein reacts with approximately 1.5 molecules of K2HgI4 per protein subunit and that the reaction involves mercury-sulfur bond formation. Crystallographic studies using difference density maps showed that one mercury is coordinated to the sulfur of cysteine-50 as Hgl and that a second mercury is shared between two cysteine-9 sulfurs of adjacent subunits, resulting in a linear S~Hg-S coordination. Gel filtration chromatography of K2HgI4-treated hemerythrin demonstrated that the protein retains its octameric structure in solution after reacting with K2HgI4, as had been observed in the crystalline state. The roles of the cysteine-9 and-50 residues in the subunit interactions of T. dyscritum hemerythrin were determined from the effects of the bulkier sulfhydryl reagents, p-hydroxy-mercuribenzoate and /V-ethylmaleimide. Binding of either reagent to cysteine-50 resulted in the disruption of all subunit interactions and the conversion of the native octamers to monomers. Binding of either reagent to cysteine-9 caused the isolated monomers to reassociate as dimers. The critical effect of cysteine-50 modification on subunit interactions was found to be due to its proximity to the major points of subunit contact, as seen in the crystal structure of the protein. © 1979, American Chemical Society. All rights reserved.