Monomeric structure of an active form of bovine cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Response to Comment on “Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry”

“…The identity of these complexes is supported by our proteomic studies, native and denaturing gels, tandem MS experiments, and detergent extraction controls. Since publication, new studies supporting many of our findings have emerged, including studies that show cardiolipin mediating binding between the BAM complex and holotranslocon (14), dimeric ANT-1 in reconstituted membranes (15), E. coli ATP synthase bound to SecYEG (16), and relatively low populations of a complex IV dimer, relative to monomer, in amphipols (17).…”

“…We acknowledge that ANT-1 can function as a monomer; so can complex V, despite being a dimer. Moreover, a single monomer of complex III is active within a dimer (6); complex IV is functional as both a monomer and a dimer, with the monomer likely the more abundant form (1,17).…”

The road to the structure of the mitochondrial respiratory chain supercomplex

“…In their structure, Yang and co-workers found NDUFA4 to be located at the site of Complex IV dimerisation and suggest it and a cardiolipin molecule also present in their structure is displaced by the cholic acid salt used in crystallisation buffers. This conclusion is supported by the work of Shinzawa-Itohet al [41], who recently solved the X-ray structure of both oxidised and reduced forms of bovine Complex IV monomers (at 1.85 Å and 1.95 Å respectively; PDB: 6JY3 and 6JY4) using novel synthetic detergents. Although NDUFA4 is absent from their structures, they found that the activity of Complex IV is lower for the dimer than for the monomer, suggesting the latter to be the active form of the complex [41].…”

Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes

“…In the matrix, Cox5B adopts the same conformation as Cox5A ( Fig. 3), with its Nterminal domain also exhibiting the conformational shift seen compared with the X-ray structure of the bovine ortholog (35,36), which most certainly arises due to the conserved interactions between Cox5 and Cor1 observed with both Cox5 isoforms ( Fig. 3D).…”

“…Whether Hig1 type 2 proteins are specific components of those CIVs that form only III-IV SCs remains to be investigated. To date, no evidence of Hig1 proteins or of NDUFA4 has been reported in structural studies of isolated mammalian CIV (35,36), and knockdown of HIG2A in mammalian cells has been shown to cause depletion of all higher-order SCs that contain CIV (27).…”