Spectroscopic Studies of Metalloproteins and Metalloenzymes

Abstract

AbstractMetalloproteins/enzymes play important rales in cellular function by virtue of their intrinsic metal ions, Various spectroscopic methods, such as atomic absorption (AA), UV/VIS absorption, NMR, fluorescence energy transfer (FET), circular dichroism (CD) and extended X‐ray absorption fine structure (EXAFS), were applied to the studies of three Zn proteins involved in gene expression and regulation. The role of the intrinsic Zn ions of E. coli RNA polymerase (RPase), Xenopus oocyte transcription factor 111A (TFIIIA), and E1A protein of adenovirus were investigated. Various paramagnetic‐metal substituted RPases obtained by in vivo and in vitro methods were used to elucidate the spatial relationship between the metal and other active sites of RPase. Two Zn ions of RPase were involved in the substrate and template binding, in maintaining the proper conformation of active RPase, and may be coordinated with histidine and cystidine. Thus, Zn ions of RPase play catalytic, regulatory and structural roles. Two Zn ions of TFIIIA also play multiple roles in the regulation of 5S RNA synthesis; binding TFIIIA to 5S RNA and its gene, essentiality for 5S RNA gene transcription and DNA‐activated ATPase activity of TFIIIA. CD studies of apoTFIIIA, TFIIIA and its storage 7S particle, and 5S RNA indicated (1) Zn ions may not involve in the stabilization of TFIIIA, (2) substantial confomational changes of 5S RNA upon binding to TFIIIA. Two major proteins, 289 and 243 amino acids (aa), of E1A gene contain 1 and O Zn, respectively. The extra 46‐aa internal domain of 289‐aa protein which contains a “Zn finger” motif with 4 cysteine residues as ligands is required for efficient transactivation of early viral promoters.