Respiratory complexes III and IV can each bind two molecules of cytochrome c at low ionic strength

Abstract

a b s t r a c tThe transient interactions of respiratory cytochrome c with complexes III and IV is herein investigated by using heterologous proteins, namely human cytochrome c, the soluble domain of plant cytochrome c 1 and bovine cytochrome c oxidase. The binding molecular mechanisms of the resulting cross-complexes have been analyzed by Nuclear Magnetic Resonance and Isothermal Titration Calorimetry. Our data reveal that the two cytochrome c-involving adducts possess a 2:1 stoichiometry -that is, two cytochrome c molecules per adduct -at low ionic strength. We conclude that such extra binding sites at the surfaces of complexes III and IV can facilitate the turnover and sliding of cytochrome c molecules and, therefore, the electron transfer within respiratory supercomplexes.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

“…In addition, significant CSPs (Δδ avg ≥ 0.05 ppm) were detected for Lys7, Lys13, His26, Lys39, Ala43, Thr78, Lys86, Lys88, and Glu89. Interestingly, Lys8, Lys13, Lys27, Lys72, and Lys86 also experienced large CSPs, as previously reported for the interaction between WT Cc and Cc 1 (47,48).…”

“…4B). The fits of CSPs along the titration suggested that Cc 1 binds two Y48pCMF Cc molecules, as previously found with the WT Cc species (47,48) (Fig. 4C).…”

“…Using NMR spectroscopy, the dissociation constant (K D ) values for the Cc 1 -Y48pCMF Cc complex were estimated to be 0.6 and 102 μM for the proximal and distal binding sites, respectively. All of the perturbed residues, except Lys55 and Ile58, surround the heme crevice, as previously described for the interaction between WT Cc and Cc 1 (47,48) (Fig. 4D).…”

“…The newly accessible amide protons in the phosphomimetic mutant (Gly29, Gly37, Arg38, Thr40, Gln42, and Trp59) are located in the surroundings of pCMF48 and the nearby Ω G -loop, in agreement with their high mobility in the microsecond-tomillisecond timescale. 1 have a 62% overall sequence identity and similar charge distributions on their molecular surfaces (47), whereas the bovine and human CcO are evolutionarily related proteins, with 91 and 96% sequence identity and homology, respectively.…”

“…Two binding sites for human and plant Cc on plant Cc 1 have been recently reported (47,48). The proximal site is located near the heme moiety and is compatible with electron transfer, whereas the distal site lies far from the heme group and probably constitutes a local energy minimum of the encounter ensemble.…”