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Abstract:
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Mitochondria are the metabolic powerhouses of the cell, taking energy released from foodstuffs and, in a series of oxidation-reduction reactions, conserving that energy into ATP, which is used to drive most reactions in the cell. These oxidation-reduction reactions use molecular oxygen as the terminal electron acceptor. The energy released is coupled to the pumping of protons across the mitochondrial inner membrane, creating the proton motive force. Cytochrome c oxidase (COX) catalyzes the oxidation of ferrocytochrome c and the reduction of oxygen to water while concomitantly translocating protons across the inner mitochondrial membrane. The catalytic core of COX consists of three subunits. The N- terminus of subunit III (SUIII) contains three conserved histidine residues that are surface exposed and are in close proximity to the mouth of the D-channel, which takes up protons to the catalytic site, where oxygen is reduced. A triple histidine mutation in SIII of COX was created using Rhodobacter sphaeroides, a bacterial model of the mitochondrion. This mutation lacks the functional groups that could participate in the uptake of protons (H3A, H7A and H10A). The mutant COX was isolated and purified by using nickel affinity chromatography. The enzyme exhibits a similar visible absorbance spectrum as wild-type enzyme; however, SDS-PAGE shows that the isolated enzyme loses 50% of SUIII content. The mutant COX retains 35% of wild- type electron transfer activity and exhibits a phenomenon called suicide inactivation, where the enzyme slowly inactivates and denatures itself. Steady-state visible absorbance spectroscopic studies indicate that electrons accumulate at heme a in the mutant during enzymatic turnover. Though the mutation does not perturb the catalytic site; it slows electron transfer activity indirectly by slowing proton uptake through the D-channel. Taken together, these results show that the removal of the three histidine residues perturbs the entry point of protons to the D-channel. |
