The CuA protein is domain II of cytochrome c oxidase and contains the CuA site, a dinuclear copper metal binding site.  Sco is a protein family necessary for the correct assembly of CuA.  Previous work has shown that Thermus thermophilus Sco (TtSco) appears to reduce the disulfide bond found in oxidized Thermus thermophilus CuA protein (TtCuA).  The reduction is necessary since oxidized TtCuA cannot bind metal. The proposed mechanism for how TtCuA is reduced by TtSco includes the formation of an intermolecular disulfide bond between Cysteine 49 on Sco and Cysteine 153 on CuA, creating a mixed disulfide intermediate (MDI). The isolation and characterization of the MDI is possible when TtSco and TtCuA are mutated to remove one cysteine.  A label is conjugated to TtCuA and is released by the attack of TtSco.  The rate at which the mixed disulfide intermediates formed as well as the total amount of MDI formed is increased when the ration of TtSco Cysteine 49 to TtCuA Cysteine 153 is increased from 1:1 to 1.5:1.  In order to isolate the mixed disulfide intermediates, size exclusion and Ni2+- affinity chromatography are utilized.  To better understand why it is TtScocysteine 49 that forms the disulfide bonds with TtCuA, Sco mutants C53S/R48A and C53S/R48D were utilized to examine the role of Arg 48.  The rate and extent of MDI formed are then compared to TtSco Cysteine 49 without the arginine mutants.

Isolating and characterizing mixed disulfide intermediate of mutant Sco and Cu<sub>A</sub> from <i>Thermus thermophilus</i>

247th National Meeting (2014)

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Isolating and characterizing mixed disulfide intermediate of mutant Sco and CuA from Thermus thermophilus

Isolating and characterizing mixed disulfide intermediate of mutant Sco and Cu_A from Thermus thermophilus