New Ligands of the Conserved Steroid Binding Site of Cytochrome c Oxidase

Abstract

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain of mitochondria and many bacteria, requires the uptake of protons for activity via two pathways, the D-path and the K-path. Among the mutants of Rhodobacter sphaeroides (Rs) CcO that inhibit proton uptake in the K-path is E101A in subunit II, which removes a key carboxyl at the entrance to the pathway and decreases the activity of the purified enzyme over 20-fold. Micromolar levels of the bile acids cholate or deoxycholate were shown to stimulate E101A activity 10-fold, and crystals of wildtype RsCcO grown in the presence of deoxycholate (Qin et al., Biochemistry 47:9931-9933, 2008) showed a single deoxycholate molecule bound close to E101 in the same location as a cholate molecule observed in bovine CcO crystal structure (PDB 1OCC). Evidence of protective effects of steroids against CcO inhibition in bilirubin neurotoxicity and in Alzheimers (Vaz et al., J. Neurochem. 112:56-65, 2010; Tillement et al., Steroids 71:725-735, 2006) suggest that this site could be physiologically relevant to regulation of CcO.Here we use the RsCcO mutant E101A as a sensitive assay to further investigate the nature of this conserved site. Activity assays show that certain other steroids (cholesterol hemisuccinate) and lipidic molecules (phytanic acid, arachidonic acid) and some detergents affect RsCcO in a manner that indicates that these ligands also bind at the same site. The studies reveal a high degree of specificity and suggest possible physiological regulators. Crystallographic and computational approaches are underway to identify additional potential ligands and to determine the physiological significance of this conserved binding site. (NIH GM26916)