A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1

Dabir, Deepa V.; Leverich, Edward P.; Kim, Sung-Kun; Tsai, Frederick D.; Hirasawa, Masakazu; Knaff, David B.; Koehler, Carla M.

doi:10.1038/sj.emboj.7601909

Cited by 148 publications

(206 citation statements)

References 45 publications

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“…The resultant reduced form of Mia40 is oxidized by a sulfhydryl oxidase Erv1 in the IMS for the next round of disulfide bond transfer (7,8). Reduced Erv1 is oxidized by cytochrome c, which is then oxidized by electron transfer to either the cytochrome oxidase complex or to cytochrome c peroxidase (12,13). The disulfide transfer reaction mediated by Mia40 is promoted by another small protein in the IMS, Hot13 (9).…”

Section: Structural Basis Of Yeast Tim40/mia40 As An Oxidative Translmentioning

confidence: 99%

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Kawano

Yamano²,

Naoé³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 Å. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

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Section: Structural Basis Of Yeast Tim40/mia40 As An Oxidative Translmentioning

confidence: 99%

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Kawano

Yamano²,

Naoé³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…In this report, we provide direct experimental evidence to show that the N-terminal Cys 30 (27). The redox potentials for the mitochondrial intermembrane space, GSH, and DTT were determined to be Ϫ255, Ϫ240, and Ϫ330 mV, respectively (31,32), and an even lower value for TCEP was predicted (33).…”

Section: Discussionmentioning

confidence: 99%

“…3A, oxygen consumption was catalyzed as soon as the WT Erv1p was added. The k cat was determined to be 1.3 Ϯ 0.1 s Ϫ1 , ϳ50% higher than that reported previously (24,27). The K m for molecular oxygen was determined to be 57 M (Fig.…”

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confidence: 84%

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Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Ang

2009

Journal of Biological Chemistry

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Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX 16 C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX 16 C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1-4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4 -6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the "small Tim" and Cox17 families (7-10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11-13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1␣ and Ero1␤) are large flavoenzymes present in the ER with...

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“…The SAGE tag for beta-glucanase gene was detected six times, the cases for peroxidase four times, and the putative peroxidase genes twice. In general, both beta-glucanases and peroxidases have been known as enzymes that mediate the defense system against abiotic stresses such as ozone, H 2 O 2 and ultraviolet radiation (Cheong et al, 2000;Dabir et al, 2007;Kim et al, 2008b;Thalmair et al, 1996). The tags for PR-10 ( Kim et al, 2003;McGee et al, 2001;Tanaka et al, 2003) were detected a total of three times, suggesting that the protein seems to make a significant contribution as a biotic stress defense system.…”

Section: Resultsmentioning

confidence: 99%

Gene Expression Profiling in Rice Infected with Rice Blast Fungus using SAGE

Kim¹,

Kim²,

Kim³

et al. 2008

The Plant Pathology Journal

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Rice blast disease, caused by the pathogenic fungus Magnaporthe grisea, is a serious issue in rice (Oryza sativa L.) growing regions of the world. Transcript profiling in rice inoculated with the fungus has been investigated using the transcriptomics technology, serial analysis of gene expression (SAGE). Short sequence tags containing sufficient information which are ten base-pairs representing the unique transcripts were identified by SAGE technology. We identified a total of 910 tag sequences via the GenBank database, and the resulting genes were shown to be up-regulated in all functional categories under the fungal biotic stress. Compared to the compatible interaction, the stress and defense genes in the incompatible interaction appear to be more up-regulated. Particularly, thaumatin-like gene (TLP) was investigated in determining the gene and protein expression level utilizing Northern and Western blotting analyses, resulting in an increase in both the gene and the protein expression level which arose earlier in the incompatible interaction than in the compatible interaction.Keywords : abiotic stress, Magnaporthe grisea, Oryza sativa, pathogenesis-related gene, rice blast, SAGE One of the most devastating diseases in rice, called rice blast, is caused by Magnaporthe grisea (Hamer and Talbot, 1998;Kim et al., 2001). The rice blast disease is a very serious and recurrent issue in rice-growing regions of the world (Talbot, 2003). The infection usually occurs on rice plant leaves where the fungal spores of M. grisea land and attach themselves using the fungal unique adhesive structure called an appressorium (Hamer et al., 1988) to penetrate rice leaf surface by building high turgor pressure (De Jong et al., 1997). Every year, the rice blast infection has the potential to affect the amount of harvested rice by which about sixty million people can be fed (Zeifler et al., 1994), generating significant economical and humanitarian issues in rice-growing areas. It has also been reported that some strains of the fungal M. grisea not only kill rice, but also attack other major cereals and grasses (Valent and Chumley, 1991).In an effort to remedy this agricultural crisis, finding the genes that are able to defend against the fungal biotic stress would be of great value. It is an obvious interest to determine which genes would be involved in the regulation and with what gene expression level in response to M. grisea infection. Transcriptomics can be used to determine the gene expression level of the messenger RNA (mRNA) in a given cell population. Transcriptomics is the study of the transcriptome, which is the set of all mRNA or transcripts produced in a population of cells. The transcriptome varies under stress conditions because all mRNA transcripts in a cell are a reflection of the genes that are being actively expressed under any stress conditions. Two technologies can be useful tools for transcriptomics. One of the technologies is microarray analysis, which utilizes labeled cDNAs hybridized to an array of DNA element...

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A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1

Cited by 148 publications

References 45 publications

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Gene Expression Profiling in Rice Infected with Rice Blast Fungus using SAGE

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