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Intramolecular electron transfer in ascorbate oxidase
OXYGEN
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DO06 INTRAMOLECULAR ELECTRON TRANSFER IN ASCORBATE OXIDASE 0. FARVERa, S. WHERLANDb, & I. PECHTC. aDepartment of General Chemistry, Royal Danish School of Pharmacy, DK2100 Copenhagen, Denmark. bDepartment of Chemistry, Washington State University, Pullmann, WA, USA. (Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel. Ascorbate oxidase (AO), an enzyme with two subunits, each carrying four copper ions, catalyzes the reduction of dioxygen to water by ascorbate. The structure shows that the copper ions are segregated into a type 1 (Tl, blue) copper site serving as the electron acceptor and a 3 copper cluster (spectroscopically identified as a type 2 copper and a type 3 copper pair) where the dioxygen is reduced. A0 provides the best system currently available to study the evolutionarily optimized process of multi-electron reduction of dioxygen. Long range (>12A) electron transfer (ET) from Tl Cu(II) to the trinuclear center, monitored by its 330nm absorbance, has been observed following the rapid bimolecular reduction of the former site by the pulse radiolytically produced CO2’ radical. The blue (61Onm) and the 330nm signals exhibited three distinct unimolecular phases at pH 5.5 and 298K (201 s-l, 20 s-l, and 2.3 s-1). The activation parameters for the fastest (AH* + g.lkJ/mol, AS*+170 J/mol-K) and slowest (AH* 6.8kJ/mol, ASf By performing complete, time -215J/mol-K) were also determined. resolved titrations of A0 with the CO2- radical, we have found that, when the 610nm and 330nm bands are approaching full reduction, the fastest intramolecular rate constant approaches 1,000 s-l. The overall amplitudes indicate that the 330nm absorbing site is preferentially reduced intramolecularly at the beginning of the titration and that one electron transferred from the Tl site to the 330nm absorbing site is adequate to eliminate the signal from the latter. We have further studied the reduction of A0 by the superoxide ion in the presence of excess dioxygen. Superoxide reduces the Tl site ca. 2 orders of magnitude more slowly than CO2-. This is followed by virtually complete reoxidation. A similar pattern is observed by monitoring the 330nm band. Kinetic analysis and simulation of these and other results will be presented and compared to previous models for A0 and lactase. ;:
A. Messeschmidt et al, J. Mol. Biol. 224, 179 (1992). 0. Farmer & I. Pecht, Proc. Natl. Acad. Sci. USA 89,8283 (1992).
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DO06 INTRAMOLECULAR ELECTRON TRANSFER IN ASCORBATE OXIDASE 0. FARVERa, S. WHERLANDb, & I. PECHTC. aDepartment of General Chemistry, Royal Danish School of Pharmacy, DK2100 Copenhagen, Denmark. bDepartment of Chemistry, Washington State University, Pullmann, WA, USA. (Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel. Ascorbate oxidase (AO), an enzyme with two subunits, each carrying four copper ions, catalyzes the reduction of dioxygen to water by ascorbate. The structure shows that the copper ions are segregated into a type 1 (Tl, blue) copper site serving as the electron acceptor and a 3 copper cluster (spectroscopically identified as a type 2 copper and a type 3 copper pair) where the dioxygen is reduced. A0 provides the best system currently available to study the evolutionarily optimized process of multi-electron reduction of dioxygen. Long range (>12A) electron transfer (ET) from Tl Cu(II) to the trinuclear center, monitored by its 330nm absorbance, has been observed following the rapid bimolecular reduction of the former site by the pulse radiolytically produced CO2’ radical. The blue (61Onm) and the 330nm signals exhibited three distinct unimolecular phases at pH 5.5 and 298K (201 s-l, 20 s-l, and 2.3 s-1). The activation parameters for the fastest (AH* + g.lkJ/mol, AS*+170 J/mol-K) and slowest (AH* 6.8kJ/mol, ASf By performing complete, time -215J/mol-K) were also determined. resolved titrations of A0 with the CO2- radical, we have found that, when the 610nm and 330nm bands are approaching full reduction, the fastest intramolecular rate constant approaches 1,000 s-l. The overall amplitudes indicate that the 330nm absorbing site is preferentially reduced intramolecularly at the beginning of the titration and that one electron transferred from the Tl site to the 330nm absorbing site is adequate to eliminate the signal from the latter. We have further studied the reduction of A0 by the superoxide ion in the presence of excess dioxygen. Superoxide reduces the Tl site ca. 2 orders of magnitude more slowly than CO2-. This is followed by virtually complete reoxidation. A similar pattern is observed by monitoring the 330nm band. Kinetic analysis and simulation of these and other results will be presented and compared to previous models for A0 and lactase. ;:
A. Messeschmidt et al, J. Mol. Biol. 224, 179 (1992). 0. Farmer & I. Pecht, Proc. Natl. Acad. Sci. USA 89,8283 (1992).