The selenocysteine-substituted blue copper center: spectroscopic investigations of Cys112SeCys Pseudomonas aeruginosa azurin.
Azurin is a small electron-transfer protein belonging to the cupredoxin family. The Cu atom is located within a trigonal plane coordinated by two histidines (His46 and His117) and a cysteine (Cys112) with two more distant ligands (Gly45 and Met121) providing axial interactions. A Cys112SeCys derivative has been prepared by expressed protein ligation, and detailed UV/vis, EPR and EXAFS studies at the Cu and Se K-edges have been carried out. Marked changes are observed between the EPR parameters of the Cys112SeCys and WT azurin derivatives, which include a 2-fold increase in A(||), a decrease in g-values, and a large increase in rhombicity of the g-tensor. The Cu-Se and Se-Cu bond lengths obtained from analysis of the Cu and Se K-EXAFS of the oxidized protein were found to be 2.30 and 2.31 A, respectively, 0.14 A longer than the Cu-S distance of the WT protein. Unexpectedly, the Cu-Se bond lengths were found to undergo only minor changes during reduction, suggesting a very similar structure in both redox states and extending the "rack" hypothesis to the Se-substituted protein.
Ralle
Martina
M
Department of Environmental & Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, Oregon 97006, USA.
Berry
Steven M
SM
Nilges
Mark J
MJ
Gieselman
Matt D
MD
van der Donk
Wilfred A
WA
Lu
Yi
Y
Blackburn
Ninian J
NJ
D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005583:Fourier Analysis; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics; D017279:Selenocysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D017279:Selenocysteine; D001400:Azurin; D003300:Copper