Important structural features of the two-subunit RsCcO. Subunit I is shown in yellow, and subunit II is shown in green. Residues of interest are shown in stick models and colored blue, except for Q142_II, which is colored magenta for clarity. Heme groups are shown in salmon. Metals are shown as spheres and colored as follows: Fe, salmon; Cu, red; Mg, blue; Cd, magenta. Two proton uptake pathways are indicated by dotted arrows, with resolved waters in the D path shown as blue spheres. One of the resolved detergent molecules (DM-decyl maltoside), corresponding to the DM at the phosphatidyl choline (PC) site in Fig. 3C, is shown in cyan, as is one of the alkyl chains corresponding to the cardiolipin (CDL) site in Fig. 3B.

Surface representations of I-II RsCcO illustrating the position and superposition of detergent/lipid molecules in Rs, Pd, and bovine CcO structures. The superpositions were identified by overlaying the structures of I-II RsCcO, I-II PdCcO (PDB ID code 1AR1), and bovine heart mitochondrial CcO (PDB ID code 1V54). (A) Molecular surface representations of I-II RsCcO, colored by relative electrostatic potential (blue, positive; red, negative) and showing the detergent molecules, maltose headgroups, and alkyl chains resolved in the structure (C, dark blue; O, red), superimposed on the LDAO detergent molecules resolved in the I-II PdCcO structure (C, green; O, red; N, blue). The lipid bilayer region is represented by two yellow dashed lines. (B) Lipid molecules in the bovine CcO structure that occupy the same sites as alkyl chains resolved in Rs/Pd structures are shown (C, yellow). Structures of RsCcO and PdCcO are colored as in A. PC, phosphatidyl choline; PG, phosphatidyl glycerol; CDL, cardiolipin; TGL, triacylglycerol. (C) Detailed view of one of the conserved lipid-binding sites, where an alkyl chain of phosphatidyl choline resolved in bovine CcO occupies the same site as detergents resolved in RsCcO and PdCcO. Surface representation of I-II RsCcO is colored by subunits (subunit I, cyan; subunit II, wheat). The decyl maltoside from RsCcO, the detergent LDAO from PdCcO, and the partial lipid molecule in bovine CcO are colored by atom type as above. The mesh representation of the resolved decyl maltoside is shown in blue. Some well conserved residues in all three structures surrounding the alkyl chain-binding site are colored dark gray.

Comparison of the resolved waters in the D proton uptake pathway in the crystal structures of I-II RsCcO and the four-subunit RsCcO. The I-II RsCcO structure is colored by atom type (C, green; O, red; N, blue), and the structure of the four-subunit RsCcO (PDB ID code 1M56) is colored gray. Waters in I-II RsCcO structure are red; waters in four-subunit RsCcO are gray. The dotted black box indicates two additional waters resolved in the I-II RsCcO. The (2F_o − F_c) difference electron density map contoured at 1.0σ is shown in blue and magenta.

The cis-peptide bond found between Q142_II and W143_II. The structure is colored by atom type (C, green; O, red; N, blue). The (2F_o − F_c) difference electron density map contoured at 1.3σ is shown in blue.

Major crystal contact region of I-II RsCcO contributed by two engineered histidine tags at the C terminus of subunit II and two cadmium ions. One RsCcO molecule is colored by atom type (C, green; O, red; N, blue); the other molecule is colored similarly but C is yellow. Cadmium is purple. The (2F_o − F_c) electron density map contoured at 1.3σ is shown in blue. The participating amino acid residues from the two protein molecules form a tetrahedral coordination around the cadmium ion as labeled in the figure. There are two identical crystal contact interactions at this interface with a quasi-2-fold rotational symmetry axis (shown as a black dot) at the center perpendicular to the plane of the paper.

Characteristics of detergent molecules resolved in the I-II RsCcO structure. (A) Two decyl maltoside detergent molecules (colored by atom type: C, green; O, red; N, blue) resolved at the interface of two RsCcO molecules (gray and wheat). The (2F_o − F_c) difference electron density map (dark blue) surrounding the decyl maltosides contoured at 1.3 σ is shown. Tyrosine and tryptophan residues (blue) were found stacking with the head groups of the two decyl maltosides. (B) One decyl maltoside resolved on another location, also at molecular interface. The (2F_o − F_c) difference electron density map (dark blue) contoured at 1.0σ is shown. Phenylalanine residue (blue) was found forming a stacking interaction with the sugar ring.

Detailed structure of the metal ligands at the active site (A) and the inhibitory cadmium-binding site (B). (A) The covalent linkage between ring atoms of Y288_I and H284_I. The (F_o − F_c) difference simulated annealing omit map is shown in blue, contoured at 4.0 σ, calculated by omitting Cu_B, H284_I, Y288_I, Fe of Heme a₃, and all residues including heme a₃ that have atoms closer than 3 Å to them. The amino acid residues are colored by atom type (C, green; O, red; N, blue), the heme group is colored light red, and the Cu_B atom is colored purple. The ligands tentatively assigned as a water bound to heme a₃-Fe and an OH⁻ group bound to Cu_B are both colored in red. (B) An inhibitory cadmium-binding site in the structure of I-II RsCcO. The (2F_o − F_c) difference electron density map contoured at 1.3σ is shown. In the current structure (colored by atom type: C, green; O, red; N, blue), cadmium (purple) is bound to E101_II and H96_II of subunit II. Binding of cadmium alters the conformations of the ligating residues compared with that found in the unliganded four-subunit RsCcO crystal structure (yellow).