Dataset Information

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase.

ABSTRACT: Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273-->Thr, Ile-280-->Thr and Gly-317-->Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (DeltaF102-F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317-->Ser had no effect on respiratory function. However, mutations Met-273-->Thr and Ile-280-->Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.

SUBMITTER: Meunier B

PROVIDER: S-EPMC1221669 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~-180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, and measurements made in a bis-tris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52, and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential are also explored as a pathway for model building.

Dataset Information

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets