Comparison of crystallographic structures and deuterium accessibility of different redox states of cytochrome c oxidase (CcO) have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the β-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway. At the entrance of the K-pathway, a conserved crystallographically-defined steroid binding site had been previously identified. Binding of diverse amphipathic molecules including detergents, fatty acids, steroids, and porphyrins affect the activity of the Rhodobacter sphaeroides CcO variant E101A, as well as the wild type and bovine enzymes. Detergent inhibition is observed for the E101A variant but may be overcome in the presence of micromolar concentrations of steroids and porphyrin analogs. Computational modeling of lauryl maltoside, bilirubin, and protoporphyrin IX into the conserved membrane site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunits I and II, including the entrance to the K-pathway, mediates competitive ligand interactions involving two overlapping sites. The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance. Physiological ligands, specific for the steroid binding site, were identified by combining three computational approaches: ROCS comparison of ligand shape and electrostatics, SimSite3D analysis of similarity to ligand binding sites in the Protein Data Bank, and SLIDE screening of small molecules by docking. Together, the results suggest several steroids, adenine and guanine nucleotides, NAD+, FAD, and phosphorylated isoprenes as top candidates for interacting at this site, along with bile acids and porphyrins. In vitro oxygen consumption assays support some of these predicted interactions. In the wild type R. sphaeroides CcO, ATP and GDP are mildly inhibitory while the steroidal deoxycholate and fusidic acid ligands are highly inhibitory. Cytochrome c titration assays indicate nucleotides inhibit CcO activity in low cytochrome c conditions, similar to the observed ATP inhibition of mammalian CcO. These finding suggest that nucleotides regulate CcO on the conserved subunit I-III core, potentially at the steroid binding site. Overall this work predicts CcO conformational changes required for catalysis, including the conformational change of the K-pathway, and describes the first report of allosteric regulation of bacterial CcO by nucleotides. These results have been used to understand allosteric regulation by restricting conformational changes, generate a two-site model for lipid and ligand-specific regulation, and propose CcO regulation by arresting the enzyme in a state which cannot produce oxygen radical byproducts.
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