Proton and metal ion-dependent assembly of a model diiron protein.
DF1 is a small, idealized model for carboxylate-bridged diiron proteins. This protein was designed to form a dimeric four-helix bundle with a dimetal ion-binding site near the center of the structure, and its crystal structure has confirmed that it adopts the intended conformation. However, the protein showed limited solubility in aqueous buffer, and access to its active site was blocked by two hydrophobic side chains. The sequence of DF1 has now been modified to provide a very soluble protein (DF2) that binds metal ions in a rapid and reversible manner. Furthermore, the DF2 protein shows significant ferroxidase activity, suggesting that its dimetal center is accessible to oxygen. The affinity of DF2 for various first-row divalent cations deviates from the Irving-Willliams series, suggesting that its structure imparts significant geometric preferences on the metal ion-binding site. Furthermore, in the absence of metal ions, the protein folds into a dimer with concomitant binding of two protons. The uptake of two protons is expected if the structure of the apo-protein is similar to that of the crystal structure of dizinc DF1. Thus, this result suggests that the active site of DF2 is retained in the absence of metal ions.
Pasternak
A
A
The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, USA.
Kaplan
J
J
Lear
J D
JD
Degrado
W F
WF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology*; D019281:Dimerization; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019159:Nonheme Iron Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D021122:Protein Subunits; D011522:Protons; D055672:Static Electricity; D013816:Thermodynamics; D014461:Ultracentrifugation
D002413:Cations, Divalent; D019159:Nonheme Iron Proteins; D021122:Protein Subunits; D011522:Protons; D007501:Iron; D019791:Guanidine; D010100:Oxygen