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Cytochrome c oxidase

Mammalian CcO has a molecular weight of about 200 kDa and contains 13 different polypeptide subunits. The three largest form the core of the enzyme and are encoded by the mitochondrial genome, whereas the remaining 10 subunits originate from nuclear DNA. Bacterial CcO are simpler in structure and have only from three to four subunits, but the sequence homology of subunits I, II, and III to the corresponding ones of mitochondrial CcO is extremely high.

Comparison of bacterial (R. sphaeroides, 1M56) and mitochondrial (1V54) oxidases. Homologous subunit are shown with similar colors: subunit I in green, subunit II in purple, subunit III in blue. Other subunits are greyed out.

Comparison of bacterial (R. sphaeroides, 1M56) and mitochondrial (1V54) oxidases. Homologous subunit are shown with similar colors: subunit I in green, subunit II in purple, subunit III in blue. Other subunits are greyed out.

At present, mitochondrial and bacterial oxidases from five organisms have been resolved with resolution up to 1.8 Å.

The list of resolved crystallographic structures of terminal oxidases
Year Type Source Resolution PDB index
1996 aa3 bovine 2.80 Å 1OCC
1998 aa3 bovine 2.30 Å 2OCC
1998 aa3-reduced bovine 2.35 Å 1OCR
1998 aa3-CO bound bovine 2.80 Å 1OCO
1998 aa3-N3 bound bovine 2.90 Å 1OCZ
2003 aa3 bovine 1.80 Å
1.80 Å
1V54
2DYR
2003 aa3-reduced bovine 1.90 Å
1.90 Å
1V55
2EIJ
2007 aa3-Cadmium bound bovine 2.10 Å 2EIK, 2EIL
2007 aa3-Zinc bound bovine 2.70 Å 2EIM, 2EIN
2009 aa3-ox. with H2O2 in BNC bovine 1.95 Å 2ZXW
2010 aa3-CO, red. at 280K bovine 2.20 Å 3AG1
2010 aa3-CO, red. at 100K bovine 1.80 Å 3AG2
2010 aa3-NO, red. at 100K bovine 1.80 Å 3AG3
2010 aa3-CN, red. at 100K bovine 2.05 Å 3AG4
1997 aa3-2 subunits P. denitrificans 2.70 Å 1AR1
1999 aa3 P. denitrificans 3.00 Å 1QLE
2009 aa3-2 subunits P. denitrificans 2.25 Å 3HB3
2002 aa3 R. sphaeroides 2.30 Å 1M56
2002 aa3-E286Q mutant R. sphaeroides 3.00 Å 1M57
2006 aa3-2 subunits R. sphaeroides 2.00 Å 2GSM
2008 aa3-2 SU, with deoxycholic acid R. sphaeroides 2.15 Å 3DTU
2009 aa3-2 SU, reduced R. sphaeroides 2.15 Å 3FYE
2009 aa3-2 SU, reduced-CN R. sphaeroides 2.20 Å 3FYI
2000 ba3 Th. thermophilus 2.40 Å 1EHK
2005 ba3 Th. thermophilus 2.30 Å 1XME
2008 ba3 Th. thermophilus ~3.4 Å 3BVD
2009 ba3-reduced (E4Q/K258R) Th. thermophilus 2.8-3.1 Å 3EH3 3EH4 3EH5
2000 bo3-quinol oxidase E. coli 3.50 Å 1FFT

Subunit I
Subunit I is the largest subunit of CcO with a molecular weight of about 60 kDa. It consists of 12 transmembrane helices without any large extramembrane domain When viewed from the top (P-side), the 12 segments of subunit I form three semicircular arcs with three pores in the centers of the arcs.
  • Pore B houses the binuclear center (heme a3 and CuB) of the oxidase and includes the proton conductive K-channel directed to the binuclear center from the N-side of the membrane.
  • Pore C retains heme a.
  • Pore A contains no cofactors but has the proton conductive D-channel as well as a plausible pathway for oxygen delivery to the binuclear site.
Top view of subunit I; only metal centers and transmembrane helices are shown. Helices form three semicircular arcs denoted A (orange), B (green), and C (blue)

Top view of subunit I; only metal centers and transmembrane helices are shown. Helices form three semicircular arcs denoted A (orange), B (green), and C (blue).

Heme a is located in pore C at a depth of about 1/3 of the membrane thickness from the P-side. The heme iron is bound to four nitrogens of the porphyrin ring and to two conserved histidines of subunit I; the bonds between the histidines and the heme iron hold the heme in the protein.

The second A-type heme is situated at about 13 Å (center-to-center distance) from heme a and is denoted as a3. Heme a3 is a high-spin heme which means that the heme has only one histidine ligand leaving one side of the heme empty and available for binding of ligands such as dioxygen, carbon monoxide, water molecules etc.

The last redox metal center of subunit I is ~5 Å away from heme a3 iron and is formed by a copper atom denoted as CuB. Together, heme a3 and CuB form the binuclear catalytic center of the oxidase responsible for binding and subsequent catalysis of oxygen.

Catalytic center of cytochrome c oxidase: high-spin heme a3, CuB, histidine-tyrosine cross-linked structure, and their ligands. Amino-acid numbering from P. denitrificans / bovine enzymes.

Catalytic center of cytochrome c oxidase: high-spin heme a3, CuB, histidine-tyrosine cross-linked structure, and their ligands. Amino-acid numbering from P. denitrificans / bovine enzymes.

In addition to heme a3 and CuB catalytic center of the oxidase contains also covalently bound histidine and tyrosine dimer. Formation of the bond between these amino acid residues could considerably reduce the pKa of the tyrosine, allowing it to participate in the oxygen reduction process by donating both the proton and the fourth electron required for the reaction, and forming a tyrosine radical.

In addition to the redox-active centers, subunit I also contains tightly bound non-redox active metal centers: a Mg2+/Mn2+ binding site at the interface between subunits I and II, and a sodium (in mitochondria) or calcium (in bacteria) binding site in a loop between helices I and II close to the P-side of the membrane.

Subunit II
Subunit II is another CcO subunit, which contains redox active cofactors. It has a molecular weight of about 27 kDa, and forms two transmembrane helices interacting with subunit I and a large C-terminal hydrophilic globular domain at the P-side of the membrane. The redox active copper center situated in the globular domain almost on the border with subunit I, conventionally referred to as CuA, is formed by two copper atoms. The CuA center in the oxidized state of the enzyme was found to be in mixed-valence configuration that can be formally represented as [Cu1.5+-Cu1.5+]. Upon reduction CuA holds an electron by sharing it between both copper atoms.

Biatomic CuA together with its ligands. P. denitrificans numbering.

Biatomic CuA together with its ligands. P. denitrificans numbering.


Subunit III
Subunit III is the biggest subunit of CcO that has no redox cofactors. It has a molecular weight of about 30 kDa, and consists of seven transmembrane helices without any extensive extramembrane domain. The helices of subunit III are arranged into two bundles. The bundles of helices are tilted with respect to each other forming a large V-shaped cleft joined at the N-side of the membrane. Subunit III might be involved in the stabilization of the mature oxidase and in ensuring correct assembly of the enzyme. In the same time, the V-shaped cleft is located at the mouth of the oxygen conducting channel and may secure a constant flux of oxygen into the catalytic center. It is also possible that the membrane-anchored cytochrome c552, which is a physiological electron donor for CcO from P. denitrificans , might use this cleft for binding and placing itself in an appropriate position for electron transfer to CuA.

Other subunits
In addition to the three core subunits, heme-copper terminal oxidases can have extra subunits. CcO from P. denitrificans has one additional subunit with a molecular weight of about 5 kDa. It has one transmembrane helix, which is in contact with all other subunits.

In mammalian CcO the three mitochondrially-encoded core subunits are supplemented by ten additional subunits, which are encoded by nuclear DNA. Seven out of ten nuclear encoded subunits consist of one transmembrane helix each, while subunits Va, Vb at the N-side and VIb at the P-side are small globular proteins. Subunit Vb contains tightly bound Zn2+.



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