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 ba3-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 O2-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the CuB atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fea3 and CuB atoms that is best modeled as peroxide. The structure of ba3-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 ba3-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3fromThermus thermophilus

“…In cytochrome ba 3 from T. thermophilus (36,37), propionate-A of heme a 3 is within hydrogen bond distance to D372 and H376, as well as to two water molecules (PDB ID code 3S8F) (38) (Fig. 1).…”

Characterization of the P IB -Type ATPases Present in Thermus thermophilus

“…Similarly, the Sco1/PrrC/SenC family of proteins has been implicated in copper acquisition by bacteria and in the assembly of copper-containing proteins, including respiratory oxygen reductases (11,23,24,59,60). Since P IB -type copper pumps have been implicated in the assembly of the copper-containing cbb 3 -type respiratory oxygen reductases (26,35), it is reasonable that the periplasmic copper chaperones present in T. thermophilus may function together with CopA and/or CopB in the assembly of the ba 3 -type and caa 3 -type copper-containing respiratory oxygen reductases present in T. thermophilus (55,61).…”

Cytochrome c 6 of Cyanobacteria and Algae: From the Structure to the Interaction

“…Such three-dimensional structures are from Paracoccus denitrificans (Koepke et al 2009;Harrenga and Michel 1999;Ostermeier et al 1997), Rhodobacter sphaeroides (SvenssonEk et al 2002;Qin et al 2006Qin et al , 2009, Rhodothermus marinus (Srinivasan et al 2005), Thermus thermophilus (Williams et al 1999;Soulimane et al 2000;Liu et al 2009;Lyons et al 2012;Tiefenbrunn et al 2011;Luna et al 2012) and Bos taurus (Tsukihara et al 1996(Tsukihara et al , 2003Yoshikawa et al 1998;Muramoto et al 2007;Suga et al 2011).…”