Structure-function relationships within cytochrome c oxidase and Complex I

NADH-dehydrogenase ubiquinol-oxidoreductase (Complex I) and cytochrome c oxidase (Complex IV) are two multi-subunit enzymes of the mitochondrial electron transport chain. Our understanding of the structural and functional aspects of these complexes is presently at different stages. We know a great deal about Complex IV, including subunit composition, role of bound phospholipids, and general aspects of electron and proton transport. The focus is now aimed at understanding the roles of subunit chemical modification, and at defining the modes of protein-protein interaction, the basis for drug-targeting aimed research. On the other hand, a crystal structure of Complex I is not yet available, and very little is known about this enzyme, especially about the role of phospholipids on Complex I structural and functional organization.;Cytochrome c Oxidase. Oxidative damage within cytochrome c oxidase mitochondrially-encoded subunits. A selectively oxidized tryptophan (Trp334) was identified within mitochondrially-encoded subunit I of cytochrome c oxidase, following reaction of its binuclear center with hydrogen peroxide. The modified residue was detected by reversed-phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry of peptides generated by enzymatic digestion of subunits I, II, and III resolved by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, with a total sequence coverage of 84%, 66% and 54%, respectively. Trp 334 is located on the surface of cytochrome c oxidase, within the mitochondrial inner membrane, at its interface with the matrix. A network of aromatic amino-acids links the distant binuclear center to the oxidized tryptophan residue. We propose that free radicals generated at the binuclear center are transferred through this network, to the terminal, surface-exposed tryptophan, where they react with oxygen to produce hydroxytryptophan.;Cytochrome c binding. The mode of interaction of cytochrome c oxidase with its substrate cytochrome c remains unclear, especially because detergentsolubilized cytochrome c oxidase can be either dimeric, monomeric or consist of a mixture of monomeric and dimeric enzyme (Musatov et al., 2000). To address this problem, sedimentation velocity analysis was used to determine the stoichiometry and affinity of binding of cytochrome c to monomeric and dimeric cytochrome c oxidase. A novel aspect of the method was the ability to assess binding to both forms of cytochrome c oxidase in the same sample. Two models are consistent with the binding data. According to the first model, monomers and the monomeric units within dimers each bind two molecules of cytochrome c; however, within the dimer, cytochrome c oxidase has a higher affinity for cyt c at both the high- and low-affinity binding sites (monomer Kd1=230 nM and Kd2=5 microM; dimer Kd1=10 nM and Kd2=1 microM). In the second model, the cytochrome c binding affinity at the two sites remains unchanged when the enzyme dimerizes; however, a new high affinity binding site emerges on dimeric cytochrome c oxidase.;Complex I. Structural studies. A high-resolution atomic structure of the enzyme is not yet available, therefore efforts are being made to elucidate the details of its structural elements by other biophysical means. We employed reversed-phase high-performance liquid chromatography to isolate Complex I subunits. With this approach we have been able to resolve 36 of the 38 nuclearly-encoded subunits of Complex I. Subunit(s) within each elution peak were positively identified by searching tandem mass spectra of tryptic peptides against the Swiss-Prot database. Reversed-phase high-performance liquid chromatography can now be used: (1) to isolate large amounts of highly purified subunits; (2) to quantify changes in subunit composition; (3) to detect chemical modification of specific subunits.;Subunit assignment to Complex I sub-domains. Previous studies have attempted to assign Complex I subunits to three enzyme sub-domains, which are generated by disruption of the enzyme in the non-denaturing detergent lauryl dimethylamino oxide, followed by anion-exchange chromatography. Following this procedure with some modifications, we analyzed the sub-domains subunit composition using reversed-phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry of tryptic digests of the respective subunits separated by "short-run" sodium dodecyl sulfate polyacrylamide-gel electrophoresis. The subunit assignments are in agreement with those reported in the literature.;Phospholipid analysis. The composition, binding sites and role of bound phospholipids on the function and/or structure of Complex I are not known. To begin to answer these questions, we first quantified the phospholipids associated with Complex I, and then with its sub-domains. Then we determined the accessibility of the phospholipids to phospholipase A 2 hydrolysis. A total of 5 cardiolipins and 5 phosphotidylethanolamines are associated with the enzyme. Two cardiolipins and 4 phosphotidylethanolamines are buried within the enzyme structure. Activity measurements of the lipid-depleted enzyme indicate that 3-4 cardiolipins are indispensable for full enzymatic activity, and that removal of these cardiolipins causes loss of 44% of Complex I activity.