Structural insights into electron transfer in caa3-type cytochrome oxidase

Abstract

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.

Figure 1. Structure of and cofactor arrangement in caa₃-oxidase

a, Structure (ribbon model). SU I/III is colored to highlight its canonical subunits: SU I, blue; SU III, blue-grey and the fusion linker, dark blue. SU IIc is colored to highlight the classical SU II (red) and the fused cytochrome c domain (green). SU IV is in yellow. The hemes are in ball and stick with the iron and copper metal centers as grey and copper spheres, respectively. Membrane boundaries are based on hydrophobic thickness calculations from the OPM server. b, Cofactor arrangement. Hemes c, a_s and a_s3, and the iron and copper ions are shown as in a. The magnesium ion is represented as a light blue sphere. Distances are shown in brown. SU I/III, IIc and IV are color coded as faded blue, red and yellow ribbons, respectively.

Figure 2. Active site, water pool and oxygen channel in caa₃-oxidase

a, b, The active site of heme a_s3 viewed from (i) the oxygen entry side and (ii) the periplasm. In a is shown a 2mFo-DFc electron density map contoured to 1σ (blue) and an anomalous difference map contoured to 5σ (red). In b is shown an mFo-DFc electron density difference map calculated without the bridging ligands contoured to 4σ (green). For clarity, the anomalous map is not shown in a(ii). c, Water pool. A water filled cavity centered on a magnesium ion is situated above heme a_s3. Coordinate bonds are shown as dashed black lines. d, The calculated oxygen channel is Y-shaped and hydrophobic. Two extremities of the channel (red) contact the apolar surface of the protein (black arrows); a third contacts the binuclear centre.

Figure 3. Proton pathways in caa₃-oxidase

a, The D-pathway begins at Asp 103 and leads through a solvent filled cavity to Tyr 248. Alternative conformations for the Asp 103 side chain were modeled into electron density and are shown. The conformer pointing towards Asn 110 represents the conformation observed in other known oxidases. b, Detailed view of the YS gate, the structural equivalent in caa₃-oxidase of the classical Type A1 gating glutamate. c, The K-pathway originates at Glu 84 of SU IIc and continues up to the binuclear centre via the cross-linked His 250 - Tyr 254, by way of Lys 328. Hydrogen bonds are shown as dashed black lines in a–c. The known covalent linkage between His 250 and Tyr 254 was modeled based on separate biochemical characterization.

Figure 4. Cytochrome c/cupredoxin complex and electron transfer pathway in caa₃-oxidase

a, Structure of the cytochrome c (green) and cupredoxin (red) domains of SU IIc. b, The optimum electron transfer pathway between the heme c iron and the Cu_A center was calculated with HARLEM. Through bond and through space tunneling are shown in solid and dashed green lines, respectively. c, “Open book” view of surface complementarities between the cytochrome c and cupredoxin domains in caa₃-oxidase. Electrostatic potentials are viewed at ±10 k_bT. The linker (residues 217-235) between the two domains has been removed for clarity. Surfaces are at 25 % transparency to reveal the positions of heme c and the Cu_A centre shown in black ball and stick and copper spheres, respectively.