Linked Life Data

17295442

Source:http://linkedlifedata.com/resource/pubmed/id/17295442

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
2007-2-13
pubmed:databankReference
pubmed:abstractText
Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (<or=1.0 A). The crystal structure of oxidized M98A amicyanin exhibits a type 1 ligation geometry but with the axial ligand provided by a water, which fills the void left by the mutation of Met to Ala. The protein undergoes a reversible switch in ligation geometry when going from the aqueous to the frozen state. The visible absorption spectrum in solution is characteristic of type 1 copper, consistent with the crystal structure. On freezing, the blue color is lost, and EPR spectroscopy reveals that the copper is primarily type 2. The crystal structure of reduced M98A amicyanin exhibits an unprecedented ligation geometry in which the His95-Cu coordination is broken, and copper is left with only two ligands from His53 and Cys92 in an almost linear coordination. The replacement of Met98 with Gln yielded a type 1 copper site with increased rhombicity evident from its EPR and visible absorption spectra, and an increase in distance from Cu to the trigonal equatorial plane seen in the crystal structure. Gln98 coordinates more strongly with copper than Met, and the oxidized and reduced forms each exhibit two alternate conformers. EPR and metal analysis of oxidized M98Q amicyanin indicate that a small population of the protein contains weakly bound type 2 copper, which may be removed by washing with EDTA. These results demonstrate that the identity as well as position and rigidity of the axial ligand of the type 1 copper site has a profound influence in the uptake specificity of metal ions, protein stability, and determination of the active site geometry and its spectroscopic properties.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
46
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1900-12
pubmed:dateRevised
2007-12-3
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties.
pubmed:affiliation
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, N.I.H., Extramural