Subunit II of cytochrome oxidase

References

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase with an engineered dinuclear copper centre. Wilmanns, M., Lappalainen, P., Kelly, M., Sauer-Eriksson, E. & Saraste, M. (1995) Proc.Natl. Acad. Sci. USA 92, 11995-11999.

Evolution of cytochrome oxidase. Saraste, M., Castresana, J., Higgins, D., Lübben, M. & Wilmanns, M. (1996) in "Origin and Evolution of Biological Energy Conversion", Ed. H. Baltscheffsky, Verlag Chemie, New York, in press.

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Summary (04/96)

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Cytochrome oxidase is an integral membrane protein that catalyses the terminal reaction in aerobic respiration, reduction of oxygen to water which is coupled to the generation of a proton gradient across the membrane (Babcock & Wikström, 1992). The cytochrome oxidase superfamily has two major branches; cytochrome bo quinol oxidases (QO) use ubiquinol and cytochrome c cytochrome oxidases (CO) use cytochrome c as primary electron donor (Garcia-Horsman et al., 1994; Saraste et al., 1996). Most of the bacterial cytochrome oxidases consist of three subunits. Subunit I is localised within the membrane and contains the active centre. Subunit II is attached to the periplasmic side of the membrane and is anchored by two N-terminal helices into the membrane. This subunit functions as primary electron acceptor. Subunit II of CO contains a dinuclear copper centre, referred to CuA. In contrast, no copper site is found in subunit II of QO. The CuA binding site in CO contains two copper ions in a mixed valence [Cu(1.5)-Cu(1.5)]S=1/2 configuration (Kroneck et al., 1988; Antholine et al., 1992). The CuA binding site has been reengineered in subunit II of QO from Escherichia coli by mutating five residues that were identified as ligands of the two coppers (Van der Oost et al., 1992). The functional role of subunit III, located within the membrane adjacent to subunit I, remains unclear.

The crystal structure of the periplasmic fragment (205 residues) of subunit II of QO from Escherichia coli, referred to CyoA, has been determined at 2.5 Å resolution by multiple isomorphous replacement methods (Wilmanns et al., 1995). Furthermore, the 3D structure of a shorter fragment (183 residues) with the re-engineered CuA binding site, referred to purple-CyoA because of its visible spectrum (Kelly et al., 1993), was solved at 2.3 Å resolution using the wt-structure as template (Wilmanns et al., 1995). At the same time the crystal structures of the entire cytochrome oxidases from Paracoccus denitrificans with 3 subunits (Iwata et al., 1995) and from bovine heart with 13 subunits (Tsukihara et al., 1995) were solved at 3.0 and 2.8 Å resolution, respectively. The fold of the soluble fragment of subunit II seems to be very similar in all three structures. A systematic comparison, however, has not yet been possible
since the co-ordinates of the other cytochrome oxidase structures are not yet available.

CyoA is folded as antiparallel b-sandwich with 11 b-strands followed by three C-terminal a-helices (Figure 3). The distance between the two copper ions in the CuA binding site is about 2.5 Å. Two cysteines function as bridging ligands of the copper ions, and two histidines are terminal ligands of each of the two copper ions. The overall arrangement of the CuA centre is highly symmetrical, which is consistent with various spectroscopic, mutagenesis and theoretical data on the CuA binding site (Malmström & Aasa, 1993; Kelly et al., 1993; Farrar et al., 1995). The ligand arrangement is that of the proposed "bridging model" (Farrar et al., 1995; Henkel et al., 1995) as opposed to the "terminal model" (Blackburn et al., 1994). The structure of purple-CyoA with reduced CuA binding site shows a virtually identical arrangement of the two coppers (Djinovic, Wilmanns & Saraste, unpublished results). However, the shortest distance between each copper and the imidazole ring of each histidine ligand has increased from about 1.9 A to 2.1 Å due to increased ion radius of Cu+1.

A shorter version of the b-sandwich fold of the subunit II of quinol oxidase was found in a variety of blue copper proteins that contain mononuclear copper binding sites (Adman, 1991). Comparisons of the overall folds and the copper binding sites have suggested common evolutionary ancestry (Adman, 1995; Saraste et al., 1996; Wilmanns & Saraste, unpublished results). Furthermore, several members of the dinitrification pathway (nitrite reductase, nitric oxide reductase and nitrous oxide reductase) seem to be related to components of cytochrome oxidase. Interpretation of parts of these data by evolutionary tree analysis has led to the "early respiration hypothesis" (Castresana et al., 1994; Castresana & Saraste, 1995). This hypothesis suggests that aerobic respiration preceeded photosynthesis during evolution. There is evidence from EPR spectroscopy that nitrous oxide reductase shares the same dinuclear copper centre as that identified in subunit II of cytochrome oxidase (Scott et al., 1989; Farrar et al., 1991; Antholine et al., 1992).


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