PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper‐binding protein and may have a thiol‐disulfide oxidoreductase activity

FEBS Letters

McEwan, Alastair G³  Boetzel, Ruth²  Davy, Sharon L²  Moore, Geoffrey R²  Wood, Tania³  Lewin, Allison²  Walker, Daniel¹  Leech, Andrew¹ 

[1] School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK;School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK;School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Qld 4072, Australia

关键词: PrrC;    Sco;    Cytochrome oxidase;    Photosynthesis response regulation;    Copper protein;    PrrC;    the Sco homologue from R. sphaeroides;    ht-PrrC;    his-tagged recombinant PrrC;    rPrrC;    recombinant PrrC starting at residue 25 of PrrC and with a five amino acid extension at the N-terminus;    apo-PrrC;    metal-free rPrrC;    ESI-MS;    electrospray ionisation mass spectrometry;    NTA;    nitrilotriacetic acid;    DTNB;    5;    5′-dithio-bis(2-nitrobenzoic acid);    DTT;    dithiothreitol;   

DOI  :  10.1016/S0014-5793(02)02532-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

PrrC from Rhodobacter sphaeroides provides the signal input to a two-component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052-2062; Oh, J.-I. and Kaplan, S. (2000) EMBO J. 19, 4237-4247). It is also a homologue of eukaryotic Sco proteins and each has a C-x-x-x-C-P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the Cu_A centre of respiratory cytochrome oxidase. Overexpression and purification of a water-soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni²⁺ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni²⁺ and formation of renatured metal-free rPrrC (apo-PrrC), Cu²⁺ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV-visible spectrum, having absorbance with a λ _max of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal-free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O₂ sensitivity are involved in copper binding in reduced PrrC since the same copper-loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol-disulfide oxidoreductase function and a copper-binding role.

【 授权许可】

Unknown