The K-path entrance in cytochrome c oxidase is defined by mutation of E101 and controlled by an adjacent ligand binding domain

Abstract

Three mutant forms of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) were created to test for multiple K-path entry sites (E101W), the existence of an “upper ligand site” (M350 W), and the nature and binding specificity of the “lower ligand site” (P315W/E101A) in the region of a crystallographically-defined deoxycholate at the K-path entrance. The effects of inhibitory and stimulatory detergents (dodecyl maltoside and Tween20) on these mutants are presented, as well as competition with other ligands, including the potentially physiologically relevant ligands cholesterol and retinoic acid. Ligands are shown to be able to compete with natural lipids to affect the activity of membrane-bound RsCcO. Results point to a single K-path entrance site at E101, with a single ligand binding pocket proximal to the entrance. The affinity of this pocket for amphipathic ligands is enhanced by removal of the E101 carboxyl and blocked by substituting a tryptophan in this area. A new crystal structure of the E101A mutant of RsCcO is presented that illustrates the structural basis of these results, showing that the loss of the E101 carboxyl creates a more hydrophobic groove consistent with altered ligand affinities.

Monomeric structure of an active form of bovine cytochrome c oxidase

“…In structural terms, the K-pathway of monomeric bovine CcO matches that of the Rhodobacter sphaeroides and Paracoccus denitrificans enzymes ( Rs CcO and Pd CcO, respectively), both of which are A-type members of the HCOR superfamily. In the glutamate residue variant at the entry point of the Rs CcO K-pathway, enzymatic activity increases in the presence of bile salts such as cholate or amphipathic carboxylates and decreases in the presence of cholesterol (36–38). In addition, in the structures of Rs CcO and Pd CcO, helix II of subunit II containing the K-pathway entry point has a higher temperature factor than other parts ( SI Appendix , Fig.…”

Interaction of Cytochrome C Oxidase with Steroid Hormones

“…Ferguson-Miller and collaborators [ 14 ] pointed out close proximity of the BABS to the entry of the K-proton channel and proposed that the amphipathic ligands may impair proton delivery to the oxygen-reducing site via the K-pathway. This proposal was supported by strong modulation of the BABS ligand effects on the activity of CcO by mutations in residue E101 in Subunit II that is thought to be the entry point for protons in the K-pathway, see Hiser et al [ 16 ]. The inhibition of intra-molecular electron transfer from heme a to heme a 3 by estradiol observed in our work is consistent with inhibition of the K-channel.…”

“…A promising new venue for research of CcO regulation has been provided by recent works of Ferguson-Miller and collaborators. In a series of papers [ 12 , 13 , 14 , 15 , 16 ], the Michigan group described a conserved bile acid binding site (BABS) in the crystal structure of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides or bovine heart mitochondria. The site is located near the inner side of the membrane close to the entrance of the so-called K-proton channel and binds a variety of amphiphilic ligands of diverse nature sharing structural similarity, such as bile acids, thyroid hormones, retinoic acid and many other compounds; see Buhrow et al [ 15 ].…”

Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites

“…New budding perspectives for studies on CcO regulation have arisen from the series of works from Ferguson-Miller’s laboratory. The authors revealed a conserved hydrophobic cavity in the crystal structure of CcO from Rhodobacter sphaeroides and bovine heart mitochondria, and suggested that it could represent a binding site for small amphipathic molecules [ 9 , 10 , 11 , 12 , 13 ]. The cavity is located on the inner side of the membrane at the boundary of subunits I and II and near the mouth of proton channel K. On the X-ray structures of CcO from mitochondria and R. sphaeroides , it was occupied by the bile acid molecule (cholate or deoxycholate, respectively) used in crystallization so the cavity was called the bile acid-binding site (BABS).…”