High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment

Abstract

The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H(+) and e(-) transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba(3)-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O(2)-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu(B) atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe(a3) and Cu(B) atoms that is best modeled as peroxide. The structure of ba(3)-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba(3)-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.

Figure 1. Crystal structure of the ba₃ A120F mutant within implicit lipid bilayer.

Subunit I is shown in cyan, subunit II is shown in orange, and subunit IIa is shown in pink. Monooleins in the asymmetric unit are shown in yellow and symmetry-related lipids are shown in green, with carboxylic carbons depicted as large spheres. Hemes are shown in magenta. Peroxide, Cu²⁺ and Fe ions are shown by red, brown and cyan spheres, respectively. Red and blue dotted lines indicate periplasmic space and intracellular borders of a hydrophobic slab.

Figure 2. Six clusters of monoolein molecules (green and cyan) surround ba ₃ oxidase (ribbon structure) in crystals obtained from the lipidic cubic phase.

The copy of ba ₃ in the asymmetric unit is light blue while symmetry related molecules are shown in gray. The lipid clusters comprise 34 monooleins, 20 of which are in the asymmetric unit. Clusters 4 and 6 mediate protein-protein packing interactions in the lattice. Clusters 2 and 3 flank an extensive direct protein-protein contact on the crystallographic 2-fold axis (space group C2) that involves the entire length of the N-terminal TM domain of Subunit II (Cu_A domain). Clusters 5 and 6 are associated with the protein in the absence of lattice interactions. Cluster 5 overlaps with the region of ordered lipid and detergent binding sites in other cytochrome oxidases, and contains the unique monoolein, OLC7, which extends above the plane of the implicit bilayer (Figure 3A).

Figure 3. Superposition of lipids associated with the current ba₃ structure and lipids associated wtih three highest resolution aa₃ oxidase structures.

Ba₃ structure is shown in green (subunit I and IIa) or orange (subunit II) and aa₃ is shown in purple. Ba₃ lipids are shown in yellow, lipids of the Bos taurus structure (2DYR) in magenta, detergents of the P. denitrificans (3HB3) in blue, and detergents of the R. sphaeroides (2GSM) structure in white. Labels with an * indicate a symmetry mate. Periplasmic and cytoplasmic membrane boundaries are shown in red and blue dotted lines. (A) Close-up of lipids in cluster 5 occupying a conserved region of lipid binding in cytochrome c oxidase and the unique lipid OLC7 that extends out of the membrane, forming specific hydrogen bonds with Arg141 and Glu144. Note that the region occupied by OLC7 in ba₃ is filled by the N-terminus of subunit II in aa₃ oxidase. (B) Close-up of lipids in cluster 1 occupying a conserved region of lipid binding throughout the cytochrome c oxidases. (C) Close-up of a region near cluster 2 that is conserved for lipid binding in aa₃-type oxidases, but not in ba₃. Note that the lipid binding region of the aa₃ oxidases is occupied by the end of helix 12 of subunit II of ba₃ oxidase (shown in orange), while the equivalent helix in aa₃-type oxidases (shown in purple) does not extend so far.

Figure 4. Internal water molecules in the high-resolution structure of ba₃ oxidase.

(A) Stick representation of main chain atoms of subunits I (blue) and II (green) of ba₃. Water molecules associated with the K-path are shown in yellow; those belonging to the unique cluster (discussed in the text) are shown in cyan; the two water molecules that bridge from Cu_B into the Xe1 site in the oxygen channel are shown in light purple; and interior waters not thought to have a functional role are shown in gray. Heme-a₃ and heme-b are shown in blue sticks, Cu_A atoms are shown as dark purple spheres; Cu_B is an orange sphere, and the peroxo dianion is shown as red spheres. The secondary OH group of the gernalygeranyl side chain of heme-a₃ is shown in green. (B) Close-up view of the internal water cluster (cyan) and K-path. Side-chains of residues of the K-path are shown in purple with a dot surface, and heme-a₃ also has a dot surface. The green sphere in the center corresponds to the secondary OH group of the gernalygeranyl side chain of heme-a₃, which also participates in the K-path.

Figure 5. Electron density around active site.

2F_o-F_c electron density is shown in green mesh at 1.5σ. The unbiased F_o-F_c difference density is shown in blue at 3.5σ.