Physico-chemical properties of molten dimer ascorbate oxidase.
The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.
Nicolai
Eleonora
E
INFM, University of Rome, Tor Vergata, Italy.
Di Venere
Almerinda
A
Rosato
Nicola
N
Rossi
Antonello
A
Finazzi Agro'
Alessandro
A
Mei
Giampiero
G
D001204:Ascorbate Oxidase / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D019281:Dimerization; D006874:Hydrostatic Pressure; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism
D010940:Plant Proteins; D003300:Copper; D001204:Ascorbate Oxidase; D014357:Trypsin