Loop-contraction mutagenesis of type 1 copper sites.
The shortest known type 1 copper binding loop (that of amicyanin, Ami) has been introduced into three different cupredoxin beta-barrel scaffolds. All of the loop-contraction variants possess copper centers with authentic type 1 properties and are redox active. The Cu(II) and Co(II) sites experience only small structural alterations upon loop contraction with the largest changes in the azurin variant (AzAmi), which can be ascribed to the removal of a hydrogen bond to the coordinating thiolate sulfur of the Cys ligand. In all cases, loop contraction leads to an increase in the pK(a) of the His ligand found on the loop in the reduced proteins, and in the pseudoazurin (Paz) and plastocyanin (Pc) variants the values are almost identical to that of Ami ( approximately 6.7). Thus, in Paz, Pc, and Ami, the length of this loop tunes the pK(a) of the His ligand. In the AzAmi variant, the pK(a) is 5.5, which is considerably higher than the estimated value for Az (<2), and other controlling factors, along with loop length, are involved. The reduction potentials of the loop-contraction variants are all lower than those of the wild-type proteins by approximately 30-60 mV, and thus this property of a type 1 copper site is fine-tuned by the C-terminal loop. The electron self-exchange rate constant of Paz is significantly diminished by the introduction of a shorter loop. However, in PcAmi only a 2-fold decrease is observed and in AzAmi there is no effect, and thus in these two cupredoxins loop contraction does not significantly influence electron-transfer reactivity. Loop contraction provides an active site environment in all of the cupredoxins which is preferable for Cu(II), whereas previous loop elongation experiments always favored the cuprous site. Thus, the ligand-containing loop plays an important role in tuning the entatic nature of a type 1 copper center.
Yanagisawa
Sachiko
S
School of Natural Sciences, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, UK.
Dennison
Christopher
C
D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010970:Plastocyanin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D002413:Cations, Divalent; D008667:Metalloproteins; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; D010970:Plastocyanin