A conserved steroid binding site in cytochrome C oxidase

Abstract

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

Figure 1

Rescuing effect of cholate (red) and deoxycholate (black) on the activity of E101_IIA mutant RsCcO. The steady-state molecular activities of the detergent-solubilized RsCcO were assayed as described in detail in the Supporting Information.

Figure 2

Deoxycholate resolved near the K path entrance of RsCcO. (A) Overview of the structure of RsCcO containing catalytic core subunits I (green) and II (cyan). The metal centers are shown as spheres (Cu, orange; Fe, red; Mg, blue; Ca, yellow; Cd, wheat). The heme groups are colored by atom type. The two proton uptake channels, D and K paths, are indicated by the black dotted lines. The water molecules within the pathways are shown as red spheres, and some of the key residues are shown as sticks. Note that the resolved deoxycholate molecule (dochl, red sticks) is located near the K path entrance. A detergent molecule, decyl maltoside (DM, blue and red sticks), is also resolved nearby. (B) The 2F_o − F_c difference electron density map (blue) contoured at the 1.0σ level. Residues E101_II and H96_II are colored by atom type, and Cd is colored yellow (PDB entry 3DTU).

Figure 3

Comparison of the conserved steroid binding sites near the K path entrance of CcO. (A) Detailed binding interactions between the deoxycholate and residues in RsCcO (subunit I, green; subunit II, cyan), including those that comprise the K path. Dochl and DM are shown as sticks and colored the same as in Figure 2. Residues that interact with the dochl molecule are shown in sticks and colored by atom type. Cd is shown as a wheat sphere. Part of the hydroxyl farnesyl tail of heme a₃ is shown as purple sticks. (B) Comparison of the binding of the steroid molecules between RsCcO and bovine CcO. Subunits I and II of both structures are represented in the same way as in panel A. Subunit VIa from a neighboring CcO molecule in the bovine CcO structure is colored wheat. Dochl resolved in the RsCcO structure is shown as red sticks. The cholate molecule (chl) found in the bovine CcO structure is shown in sticks and colored by atom type (blue and red). Part of the phosphatidylethanolamine molecule (PE) resolved in the bovine CcO structure is shown as yellow sticks. Some residues from both structures that interact with the steroid molecule are shown as sticks. The structure of RsCcO with dochl is labeled in black, while that of bovine CcO with chl is labeled in blue.