Cytochrome c Oxidase Biogenesis and Metallochaperone Interactions: Steps in the Assembly Pathway of a Bacterial Complex.

Sonja Schimo,Bernd Ludwig,Klaas M. Pos,Ilka Wittig,Alessandro Giuffrè

doi:10.1371/journal.pone.0170037

Abstract

Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.

Highlights

The heme aa3-type cytochrome c oxidase (COX) is the terminal electron acceptor in the respiratory chain of mitochondria and many bacteria
COX assembly intermediates and interacting metallochaperones detected by immunostaining Membranes from P. denitrificans, prepared from wildtype (WT) and deletion strain cells, were solubilized with the mild detergent digitonin and separated in the first dimension by blue native (BN)-polyacrylamide gel electrophoresis (PAGE), ensuring the preservation of weak protein-protein-interactions
Defects in the biogenesis pathway of mitochondrial COX are associated with severe respiratory deficiencies

Summary

Introduction

The heme aa3-type cytochrome c oxidase (COX) is the terminal electron acceptor in the respiratory chain of mitochondria and many bacteria. It catalyzes the transfer of electrons from cytochrome c to molecular oxygen, a reaction that is coupled to the translocation of protons across the membrane. It contributes to the generation of a proton gradient, which is subsequently used to drive ATP synthesis [1, 2]. The mitochondrial enzyme consists of up to 13 different subunits (SU), with three highly conserved core SU I-III [3, 4] which are encoded by the organellar genome and represent the minimal functional entity in most bacterial oxidases as well. As the main catalytic player, COX SUI houses both the heme moieties (a and a3) and a PLOS ONE | DOI:10.1371/journal.pone.0170037 January 20, 2017

Methods

Results

Conclusion