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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 dis-plays such an intermediate along its unfolding pathway. | ễFEBS Journal Physico-chemical properties of molten dimer ascorbate oxidase 1 I Al I 1_2 A I I l-s 1.2 H I l .1.2 A. II l 2 Eleonora Nicolai Almerinda Di Venere Nicola Rosato Antonello Rossi Alessandro Finazzi Agro 2 and Giampiero Mei1 2 1 INFM University of Rome Tor Vergata Italy 2 Department of ExperimentalMedicine and BiochemicalSciences University of Rome Tor Vergata Italy Keywords conformationalchange dimeric intermediate high pressure protein compressibility protein folding Correspondence G. Mei Department of Experimental Medicine and Biochemical Sciences University of Rome Tor Vergata Via Montpellier 1 00133 Rome Italy Fax 39 06 72596468 Tel 39 06 72596460 E-mail mei@med.uniroma2.it Received 9 August 2006 revised 26 September 2006 accepted 27 September 2006 doi 10.1111 j.1742-4658.2006.05515.x 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 108 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 AV w -200 mL-mol-1 by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to
