Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Katzen, Federico; Beckwith, Jon

doi:10.1073/pnas.1334136100

Cited by 42 publications

(42 citation statements)

References 25 publications

(47 reference statements)

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“…The exact position of these cysteines, whether embedded or in proximity to the stromal or luminal sides, cannot be resolved by our topological experiments. The function of the within-membrane cysteines in CCDA is not really known, but functional dissection of the related bacterial DsbD/DipZ transporter has demonstrated that the redox active cysteines face the cytoplasmic side of the membrane (50). These cysteines have the potential to form a disulfide bond, an indication of their physical vicinity, and are key residues in the relay of reducing equivalents originating from the cytoplasm (50).…”

Section: Discussionmentioning

confidence: 99%

“…The function of the within-membrane cysteines in CCDA is not really known, but functional dissection of the related bacterial DsbD/DipZ transporter has demonstrated that the redox active cysteines face the cytoplasmic side of the membrane (50). These cysteines have the potential to form a disulfide bond, an indication of their physical vicinity, and are key residues in the relay of reducing equivalents originating from the cytoplasm (50). It is likely that conserved cysteines in CCDA are also facing the stromal side of the thylakoid membrane and operate in a similar manner .…”

Section: Discussionmentioning

confidence: 99%

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A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome bf Complex in Arabidopsis Chloroplasts

Page¹,

Hamel²,

Gabilly³

et al. 2004

Journal of Biological Chemistry

101

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The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b 6 f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.The c-type cytochromes are a virtually ubiquitous yet rather structurally diverse group of hemoproteins that reside on the so-called p-side 1 of the energy-transducing membrane systems where they function typically as electron carriers (1, 2). The c-type cytochromes are defined by the occurrence of heme covalently attached to the polypeptide via two thioether linkages between the vinyl groups of heme and the cysteine sulfhydryls in the apocytochrome (1, 3). A CXXCH 2 sequence in the apocytochrome provides the thiols for formation of the thioether bonds, and the imidazole of the histidine residue serves as one of the axial ligands of the heme. Remarkably, three distinct assembly pathways, referred to as systems I, II, and III, have emerged through genetic analysis of c-type cytochrome maturation in bacteria, chloroplasts, and mitochondria (for review, see Refs. 1 and 4 -7), an unexpected finding for what appears on the surface to be a rather simple chemical reaction (i.e. the addition of apocytochrome cysteine thiols to the vinyls of heme). Each system can be distinguished on the basis of a sequence relationship of specific assembly factors. Yet, there is an underlying assumption that the three systems are united by common biochemical requirements for holocytochrome c biogenesis (for review, see Refs. 2, 4, and 8). For instance, the need for reducing conditions appears to be inherent to the chemistry of thioether bond formation (4). Both substrates, apocytochrome c sulfhydryls and heme, need to be maintained reduced prior to the ligation of heme as indicated from in vitro and in o...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome bf Complex in Arabidopsis Chloroplasts

Page¹,

Hamel²,

Gabilly³

et al. 2004

Journal of Biological Chemistry

101

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“…The electron transfer pathway from Trx1 to DsbC through DsbD (␤3␥3␣ domain) was determined by detecting the mixed disulfide complexes, Trx1-DsbD␤ and DsbD␣-DsbC, and by assessing the oxidation state of each domain when one of the other domains was missing (11). Our studies showed that in the absence of electron transfer from thioredoxin the DsbD␤ domain is found with its two cysteines joined in a disulfide bond (11,12). Thus, DsbD presents the first case of a protein that uses redox-active cysteines to transfer electrons across a membrane and that (at one stage of its activity) contains a disulfide bond in a membrane-embedded domain.…”

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confidence: 94%

Mutations of the Membrane-Bound Disulfide Reductase DsbD That Block Electron Transfer Steps from Cytoplasm to Periplasm in Escherichia coli

Cho

Beckwith

2006

J Bacteriol

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The cytoplasmic membrane protein DsbD keeps the periplasmic disulfide isomerase DsbC reduced, using the cytoplasmic reducing power of thioredoxin. DsbD contains three domains, each containing two reactive cysteines. One membrane-embedded domain, DsbD␤, transfers electrons from thioredoxin to the carboxyterminal thioredoxin-like periplasmic domain DsbD␥. To evaluate the role of conserved amino acid residues in DsbD␤ in the electron transfer process, we substituted alanines for each of 19 conserved amino acid residues and assessed the in vivo redox states of DsbC and DsbD. The mutant DsbDs of 11 mutants which caused defects in DsbC reduction showed relatively oxidized redox states. To analyze the redox state of each DsbD domain, we constructed a thrombin-cleavable DsbD (DsbD TH ) from which we could generate all three domains as separate polypeptide chains by thrombin treatment in vitro. We divided the mutants with strong defects into two classes. The first mutant class consists of mutant DsbD␤ proteins that cannot receive electrons from cytoplasmic thioredoxin, resulting in a DsbD that has all six of its cysteines disulfide bonded. The second mutant class represents proteins in which the transfer of electrons from DsbD␤ to DsbD␥ appears to be blocked. This class includes the mutant with the most clear-cut defect, P284A. We relate the properties of the mutants to the positions of the amino acids in the structure of DsbD and discuss mechanisms that would interfere with the electron transfer process.Extracytoplasmic compartments, including the external milieu, are oxidative environments in both eukaryotic and prokaryotic cells. Many proteins in these environments contain disulfide bonds as stable parts of their structures. The formation of structural disulfide bonds depends on enzymes present in certain of the extracytoplasmic compartments. In the periplasm of Escherichia coli, the enzyme DsbA acts as an oxidant to promote disulfide bond formation in secreted or membrane proteins via a thiol-disulfide exchange reaction (1). However, the formation of disulfide bonds in protein substrates by DsbA is not perfect, and occasionally nonnative pairs of disulfide bonds are introduced into target proteins. This "misoxidation" occurs most commonly in those proteins which in their native states contain disulfide bonds between cysteines that follow nonsequentially in the amino acid sequence (3). Thus, bacteria (and eukaryotes as well) require a second enzyme, a periplasmic protein disulfide isomerase, which can correct nonnative disulfide bonds in target proteins. In E. coli, this enzyme is DsbC (15, 22, 30).DsbC, which also exhibits chaperone activity (4), is thought to recognize proteins that contain nonnative disulfide bonds because they are misfolded. These bonds are attacked by one of the active-site cysteines of DsbC, presumably leading to an intermediate, mixed disulfide between DsbC and the substrate. The rearrangement of disulfide bonds in the substrate can occur either by resolution of the mixed disulfide, involving a...

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“…In E. coli, DsbD is a inner membrane-bound protein that transfers electrons to DsbC, a disulfide isomerase that repairs nonnative disulfide bonds in the periplasm (Rietsch et al, 1997) and DsbE, a thioredoxin protein essential to cytochrome c maturation (Stirnimann et al, 2005). The DsbD knockout is extremely copper sensitive, which is not surprising, since copper as a redox metal catalyzes disulfide bonds quite readily (Katzen and Beckwith, 2003), and the entire pathway is thought to be involved in copper resistance and potentially other oxidative stressors (Hiniker et al, 2005). Indeed, in Synechocystis PCC6803 the orthologous operon (sll0685-sll0688) is transcriptionally upregulated in response to hydrogen peroxide stress, osmotic stress and DCMU ((3-(3,4-dichlorophenyl)-1,1-dimethylurea)) (Singh et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Genomic island genes in a coastal marine Synechococcus strain confer enhanced tolerance to copper and oxidative stress

et al. 2013

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Highly variable regions called genomic islands are found in the genomes of marine picocyanobacteria, and have been predicted to be involved in niche adaptation and the ecological success of these microbes. These picocyanobacteria are typically highly sensitive to copper stress and thus, increased copper tolerance could confer a selective advantage under some conditions seen in the marine environment. Through targeted gene inactivation of genomic island genes that were known to be upregulated in response to copper stress in Synechococcus sp. strain CC9311, we found two genes (sync_1495 and sync_1217) conferred tolerance to both methyl viologen and copper stress in culture. The prevalence of one gene, sync_1495, was then investigated in natural samples, and had a predictable temporal variability in abundance at a coastal monitoring site with higher abundance in winter months. Together, this shows that genomic island genes can confer an adaptive advantage to specific stresses in marine Synechococcus, and may help structure their population diversity.

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Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

Cited by 42 publications

References 25 publications

A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome bf Complex in Arabidopsis Chloroplasts

A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome bf Complex in Arabidopsis Chloroplasts

Mutations of the Membrane-Bound Disulfide Reductase DsbD That Block Electron Transfer Steps from Cytoplasm to Periplasm in Escherichia coli

Genomic island genes in a coastal marine Synechococcus strain confer enhanced tolerance to copper and oxidative stress

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