Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly.

Cytochrome c oxidase (COX) assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia, and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here, we show that loss of COA7 blocks complex IV assembly after the initial step where the COX1 module is built, progression from which requires the incorporation of copper and addition of the COX2 and COX3 modules. The crystal structure of COA7, determined to 2.4 Å resolution, reveals a banana-shaped molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds. COA7 interacts transiently with the copper metallochaperones SCO1 and SCO2 and catalyzes the reduction of disulfide bonds within these proteins, which are crucial for copper relay to COX2. COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. We therefore propose that COA7 is a heme-binding disulfide reductase for regenerating the copper relay system that underpins complex IV assembly.

Structural and Functional Investigation of the Periplasmic Arsenate-Binding Protein ArrX from Chrysiogenes arsenatis.

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO43-) as the terminal electron acceptor, where it is reduced to arsenite (AsO33-) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.

What Role Does COA6 Play in Cytochrome C Oxidase Biogenesis: A Metallochaperone or Thiol Oxidoreductase, or Both?

Complex IV (cytochrome c oxidase; COX) is the terminal complex of the mitochondrial electron transport chain. Copper is essential for COX assembly, activity, and stability, and is incorporated into the dinuclear CuA and mononuclear CuB sites. Multiple assembly factors play roles in the biogenesis of these sites within COX and the failure of this intricate process, such as through mutations to these factors, disrupts COX assembly and activity. Various studies over the last ten years have revealed that the assembly factor COA6, a small intermembrane space-located protein with a twin CX9C motif, plays a role in the biogenesis of the CuA site. However, how COA6 and its copper binding properties contribute to the assembly of this site has been a controversial area of research. In this review, we summarize our current understanding of the molecular mechanisms by which COA6 participates in COX biogenesis.

Human glutaredoxin-1 can transfer copper to isolated metal binding domains of the P1B-type ATPase, ATP7B.

Intracellular copper (Cu) in eukaryotic organisms is regulated by homeostatic systems, which rely on the activities of soluble metallochaperones that participate in Cu exchange through highly tuned protein-protein interactions. Recently, the human enzyme glutaredoxin-1 (hGrx1) has been shown to possess Cu metallochaperone activity. The aim of this study was to ascertain whether hGrx1 can act in Cu delivery to the metal binding domains (MBDs) of the P1B-type ATPase ATP7B and to determine the thermodynamic factors that underpin this activity. hGrx1 can transfer Cu to the metallochaperone Atox1 and to the MBDs 5-6 of ATP7B (WLN5-6). This exchange is irreversible. In a mixture of the three proteins, Cu is delivered to the WLN5-6 preferentially, despite the presence of Atox1. This preferential Cu exchange appears to be driven by both the thermodynamics of the interactions between the proteins pairs and of the proteins with Cu(I). Crucially, protein-protein interactions between hGrx1, Atox1 and WLN5-6 were detected by NMR spectroscopy both in the presence and absence of Cu at a common interface. This study augments the possible activities of hGrx1 in intracellular Cu homeostasis and suggests a potential redundancy in this system, where hGrx1 has the potential to act under cellular conditions where the activity of Atox1 in Cu regulation is attenuated.

Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6.

Assembly factors play key roles in the biogenesis of many multi-subunit protein complexes regulating their stability, activity, and the incorporation of essential cofactors. The human assembly factor Coa6 participates in the biogenesis of the CuA site in complex IV (cytochrome c oxidase, COX). Patients with mutations in Coa6 suffer from mitochondrial disease due to complex IV deficiency. Here, we present the crystal structures of human Coa6 and the pathogenic W59CCoa6-mutant protein. These structures show that Coa6 has a 3-helical bundle structure, with the first 2 helices tethered by disulfide bonds, one of which likely provides the copper-binding site. Disulfide-mediated oligomerization of the W59CCoa6 protein provides a structural explanation for the loss-of-function mutation.

High-resolution crystal structure of the reduced Grx1 from Saccharomyces cerevisiae.

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P212121 and its structure solution and refinement to 1.22 Šresolution are reported. To study the structure-function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27-Cys30 active site which accompany alterations in the redox status of the protein.