Dataset Information

Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

ABSTRACT: The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1? mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

SUBMITTER: Bareth B

PROVIDER: S-EPMC4865315 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

Bareth Bettina B Nikolov Miroslav M Lorenzi Isotta I Hildenbeutel Markus M Mick David U DU Helbig Christin C Urlaub Henning H Ott Martin M Rehling Peter P Dennerlein Sven S

Molecular biology of the cell 20160330 10

The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly proce ...[more]

PMID: 27030670

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Project description:CuA is a dinuclear mixed-valence center located in subunit 2 of the ba(3)-type cytochrome oxidase from Thermus thermophilus. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of CuA formation from inorganic ions. The formulation of the CuA center as an electron-delocalized Cu(1.5)-Cu(1.5) system implicates both Cu(II) and Cu(I) states in the metalation process. In earlier work we showed that selenomethionine (SeM) substitution of the coordinated M160 residue provided a ligand-directed probe for studying the copper coordination environment via the Se XAS signal, which was particularly useful for interrogating the Cu(I) states where other spectroscopic probes are absent. In the present study we have investigated the formation of mixed-valence CuA and its M160SeM derivative by stopped-flow UV-vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu(I) CuA has been studied by XAS alone. Our results establish the presence of previously undetected mononuclear intermediates and show important differences from the metalation reactions of purple CuA azurin. XAS spectroscopy at Cu and Se edges has allowed us to extend mechanistic inferences to formation of the di-Cu(I) state which may be more relevant to biological CuA assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems and that the dominant intermediate along the pathway to mixed-valence is a new green species with λ(max) = 460 nm. This intermediate has been isolated in a homogeneous state and shown to be a mononuclear Cu(II)-(His)(Cys)(2) species with no observable Cu(II)-(Met) interaction. Reduction with dithionite generates its Cu(I) homologue which is again mononuclear but now shows a strong interaction with the Met160 thioether. The results are discussed within the framework of the "coupled distortion" model for Cu(II) thiolates and their relevance to biological metalation reactions of the CuA center.

Dataset Information

Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

Publications

Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

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