Basic requirements for a metal-binding site in a protein: the influence of loop shortening on the cupredoxin azurin.
The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C(112)TFPGH(117)SALM(121) to C(112)TPH(115)PFM(118) (the native loop from the cupredoxin amicyanin) and also to C(112)TPH(115)PM(117). The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C(112)TPH(115)PM(117) loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C(112)TPH(115)PFM(118) loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.
Li
Chan
C
Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom.
Yanagisawa
Sachiko
S
Martins
Berta M
BM
Messerschmidt
Albrecht
A
Banfield
Mark J
MJ
Dennison
Christopher
C
D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D008024:Ligands; C050107:cupredoxin; D001400:Azurin; D003300:Copper