X-ray absorption spectroscopic studies of metalloproteins: Mecury-substituted blue copper proteins, the MerR metalloregulatory protein, and phthalate dioxygenase.

Abstract

X-ray absorption spectroscopy has been used to characterize structurally the metal sites in metalloproteins involved in electron transfer, dioxygen activation, and regulation of gene expression. X-ray absorption studies on the Hg-substituted "blue" copper proteins plastocyanin, azurin, laccase, and stellacyanin confirm that Hg is selectively substituted in the "blue" site of laccase and reveal that the structure of this site is similar to other "blue" copper proteins. The structural properties of the "blue" site are essentially the same whether copper or mercury are bound. MerR, the biological sensor which activates the mer operon to express bacterial mercury resistance, was found to have a three-coordinate HgS$\sb3$ stoichiometry, with Hg-S distances of 2.43 A. The thermodynamic stability of this trigonal site may account for the affinity of MerR for Hg(II) and its selectivity for Hg(II). Both the Rieske-like cluster and the mononuclear iron site of Psuedomonas cepacia phthalate dioxygenase have been structurally characterized. The structure of the Rieske-cluster is indistinguishable from that of other (2Fe2S) clusters (R$\sb{\rm Fe-S}$:2.24 A for oxidized; 2.28 A, for reduced; R$\sb{\rm Fe-Fe}$:2.68 A), suggesting that its high reduction potential is due to electrostatic rather than structural factors. The mononuclear site consists exclusively of low-Z ligands, probably with more O than N. The Zn-substituted mononuclear site remains 5-coordinate (R$\sb{\rm Zn-O}$:2.02 A whether phthalate is absent or present. The Co- and native Fe-containing mononuclear site appear to change from 6- to 5-coordinate when phthalate is bound as indicated by the change in average bond length. (2.12 A to 2.06 A for R$\sb{\rm Co-O}$, and 2.11 A to 2.07 A for R$\sb{\rm Fe-O}$). A water or hydroxide ligand of the protein may be displaced because of the change of hydrophobicity on phthalate binding.