The Post-Translational Modification in Cytochrome c Oxidase Is Required To Establish a Functional Environment of the Catalytic Site

Abstract

Mutation of tyrosine-288 to a phenylalanine in cytochrome c oxidase from Rhodobacter sphaeroides drastically alters its properties. Tyr-288 lies in the CuB-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 CuB. In the Y288F mutant enzyme, the "as-isolated" preparation is a mixture of reduced cytochrome a and oxidized cytochrome a3. The cytochrome a3 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 CuB, thereby preventing nonfunctional ligands from coordinating to the heme.

Identification of a histidine-tyrosine cross-link in the active site of the cbb 3 -type cytochrome c oxidase from Rhodobacter sphaeroides

“…However, we stress that our data cannot identify the cross-linked atoms in the histidine-tyrosine dimer of cytochrome cbb 3 , although the modeled cytochrome cbb 3 structures suggest identity with the canonical oxidases in this respect (13,14). The cross-link has been proposed to maintain the structure of the active site (34,35). Studies with R. sphaeroides cytochrome aa 3 showed that in the tyrosine to phenylalanine mutant, one of the histidine ligands of Cu B might coordinate heme a 3 , Cu B is largely lost, and the optical spectrum is changed (34).…”

“…This is in contrast to the observations with cbb 3 oxidase, where the structure of the active site appears less affected (see above). As pointed out earlier (34), the cross-link may have an important role in holding the tyrosine residue in the correct orientation. In cytochrome aa 3 there is a hydrogen bonding interaction between the hydroxyl group of the tyrosine and the hydroxyl group of the hydroxyethyl farnesyl side chain of the high spin heme.…”

“…The cross-link has been proposed to maintain the structure of the active site (34,35). Studies with R. sphaeroides cytochrome aa 3 showed that in the tyrosine to phenylalanine mutant, one of the histidine ligands of Cu B might coordinate heme a 3 , Cu B is largely lost, and the optical spectrum is changed (34). This is in contrast to the observations with cbb 3 oxidase, where the structure of the active site appears less affected (see above).…”

A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis

“…To test if this tyrosine modifies the functional and the spectroscopic properties of cytochrome c oxidase, site-directed mutagenesis studies have been reported. Unfortunately, the tyrosine plays such a critical role in stabilizing the structure of the protein that the mutants were nonfunctional and the catalytic site was completely disrupted (39). Thus, no conclusions could be drawn regarding the role of this residue from the mutagenesis experiments.…”

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met–Tyr–Trp cross-linked adduct

“…It has been suggested that the positively charged sulfonium group within the adduct in KatG may raise the redox potential of the covalently bound residues [14], hindering the oxidation of Tyr229, Trp107, or both by Cmpd I and the formation of Cmpd II (plus protein radical). However, other factors may also affect redox potentials in KatG(Y229F) compared to WT KatG [77][78][79][80]. We propose that the adduct formation is important for optimization of the redox potential of the Met-Tyr-Trp adduct as a whole compared to those of the noncovalently attached side chains in KatG(Y229F) to minimize Cmpd II formation and maximize Cmpd II decay to prevent inhibition of catalase activity, while its possible role in facilitating the required proton transfer step should also be investigated.…”