Involvement of Hemerythrin Sulfhydryl Groups in Heavy Atom Binding and Subunit Interactions

Abstract

Hemerythrin is a respiratory protein found in the erythrocytes of certain marine invertebrates. Structures have.been obtained by x~ray c~ystallography for hemerythrins from Phascolopsis gouldii (P. gouldii) and:Themiste·dyscritum (!. dyscritum). Upon solving the crystal structure of T. dyscritum hemerythrin at 2.8 A resolution, two mercury binding sites were observed in the heavy atom derivative prepared by treating the protein with mercuric iodide. Since many proteins are inactivated upon binding heavy metals such as mercury, an investigation of mercury-protein interactions in this protein was undertaken. In order to understand the nature of heavy-metal protein interactions in T. dyscritum it was necessary to identify the metalloprotein ligands since neither the liga~ding ami~o acid residues nor the geometry of the mercury complexes could be identified in the crystallographic studies. · Once the amino acid sequence of_ T. dyscritum hemerythrin was determined, it was clear that both mercury binding si~es were close to the thiol groups of two cysteine residues (cysteine 9 and cysteine 50). Possible struc~ures for the mercury binding sites were identified by combining a ;knowledge of hemerythrin structure with direct evidence for mercury-thiol bond formation obtained by spectrophotometric titration of the thiol groups with mercuric iodide. The titration data showed that 1.5 atoms of mercury were bound per subunit, which 2 is consistent with one mercury bound at cysteine 50 and one mercury shared by cysteine 9's of a~jacent subunits. The mercuric iodide binding at cysteine 50 appears to be a lin~ar structure (Protein-S-Hg-I), while the·mercury bridging two cysteine 9's on adjacent subunits is better explained by a tetrahedral structure [(Protein-s) 2HgI 2 ].The relative reaction rates of the .thiol groups in hemerythrin with p-hydroxymercuribenzoate (PHMB) and N-ethylmaleimide (NEM) were investigat.ed· in order to gain information about the comparative hydrophobicity. of the environm~nts surrounding the t~o cysteines. The data showed the environment about cysteine 50 to. be hydrophobic relative to the environment about cysteine 9. This was substantiated by structural information whtch showed cysteine 50 to be buried in a subunit interface while.cysteine 9 was in a region relatively exposed 3 to the solvent.