Mutation of tyrosine-288 to a phenylalanine in cytochrome c oxidase from Rhodobacter sphaeroides drastically alters its properties. Tyr-288 lies in the Cu(B)-cytochrome a3 binuclear catalytic site and forms a hydrogen bond with the hydroxy group on the farnesyl side chain of the heme. In addition, through a post-translational modification, Y288 is covalently linked to one of the histidine ligands that is coordinated to Cu(B). In the Y288F mutant enzyme, the 'as-isolated' preparation is a mixture of reduced cytochrome a and oxidized cytochrome a3. The cytochrome α3 heme, which is largely six- coordinate low-spin in both oxidation states of the mutant, cannot be reduced by cytochrome c, but only by dithionite, possibly due to a large decrease in its reduction potential. It is postulated that the Y288F mutation prevents the post-translational modification from occurring. As a consequence, the catalytic site becomes disrupted. Thus, one role of the post-translational modification is to stabilize the functional catalytic site by maintaining the correct ligands on Cu(B), thereby preventing nonfunctional ligands from coordinating to the heme.
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