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.
Highlights
Heme/copper oxidases (HCO) represent a superfamily of enzymes found in the aerobic respiratory chain of mitochondria and bacteria that convert oxygen to water and transfer protons across membranes to form an electrochemical gradient
Overall protein structure and comparisons with lower resolution ba3 structures The wild type (WT) protein and A120F mutant of ba3 cytochrome c oxidase from T. thermophilus were crystallized in lipidic cubic phase and their structures were refined at 1.8 A (Table 1)
The enzyme was crystallized under a number of conditions; all crystallization conditions contained a high concentration of PEG400 (40–45%) that swells lipidic cubic phase (LCP) and converts it into a sponge phase [33], which is consistent with a previous hypothesis that large proteins (.50 kDa) require swelling of LCP in order to have enough room for protein molecules to move and feed into growing crystals [34]
Summary
Heme/copper oxidases (HCO) represent a superfamily of enzymes found in the aerobic respiratory chain of mitochondria and bacteria that convert oxygen to water and transfer protons across membranes to form an electrochemical gradient. Members of this family include cytochrome c and quinol oxidases that have been phylogenetically grouped into three major subfamilies. The C-type oxidases (cbb3), found in two bacterial groups, are expressed at low oxygen levels, a feature they share with some pathogenic bacteria [10]. While highly divergent in amino acid sequence, HCOs of the different types share common structural elements, suggesting a similar mechanism of action
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