Metal ion binding properties of Triticum [corrected] aestivum Ec-1 metallothionein: evidence supporting two separate metal thiolate clusters.
Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Triticum [corrected] aestivum (common wheat) early cysteine-labeled plant metallothionein (Ec-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant Ec-1 is able to bind a total of six divalent d10 metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so far.
Peroza
Estevão A
EA
Department of Chemistry, University of Zurich, 8057, Zurich, Switzerland.
Freisinger
Eva
E
D000595:Amino Acid Sequence; D002104:Cadmium / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013057:Spectrum Analysis; D014908:Triticum / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D009942:Organometallic Compounds; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D015032:Zinc