Expression and Spectroscopic Characterization of the Hydrogenosomal [2Fe-2S] Ferredoxin from the Protozoan

Vidakovic, Momcilo; Fraczkiewicz, Grazyna; Germanas, Juris P.

doi:10.1074/jbc.271.25.14734

Cited by 26 publications

(15 citation statements)

References 29 publications

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“…It has previously been observed that FdI migrates abnormally on SDS-12% PAGE as if its molecular weight were ϳ24,000 rather than 12,000 (57). Such anomalous behavior on SDS-PAGE has previously been re-ported for other small and highly charged proteins (the isoelectric point of FdI calculated from amino acid sequence is 4.03) (58). The similar purification properties and electrophoretic behaviors of FdIV and FdI suggested that they were likely to have similar charges and molecular weights.…”

Section: Resultsmentioning

confidence: 80%

Purification and Biophysical Characterization of a New [2Fe-2S] Ferredoxin from Azotobacter vinelandii, a Putative [Fe-S] Cluster Assembly/Repair Protein

Jung

Gao-Sheridan

Christiansen

et al. 1999

Journal of Biological Chemistry

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During the purification of site-directed mutant variants of2؉/؉ cluster by UV/visible, CD, and EPR spectroscopies with a reduction potential of ؊344 mV versus the standard hydrogen electrode. When overexpressed in Escherichia coli, recombinant FdIV holoprotein was successfully assembled. However, the polypeptide of the recombinant protein was modified in some way such that the apoprotein molecular mass increased by 52 Da. Antibodies raised against FdIV and EPR spectroscopy were used to examine the relative levels of FdIV and FdI in various A. vinelandii strains leading to the conclusion that FdIV levels appear to be specifically increased under conditions where another protein, NADPH:ferredoxin reductase is also up-regulated. In that case, the fpr gene is known to be activated in response to oxidative stress. This suggests that the fdxD gene and other genes in the iron-sulfur cluster assembly or repair operon might be similarly up-regulated in response to oxidative stress.

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Section: Resultsmentioning

confidence: 80%

Purification and Biophysical Characterization of a New [2Fe-2S] Ferredoxin from Azotobacter vinelandii, a Putative [Fe-S] Cluster Assembly/Repair Protein

Jung

Gao-Sheridan

Christiansen

et al. 1999

Journal of Biological Chemistry

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“…The hydrogenosomal [2Fe-2S]-ferredoxin of Trichomonas has been characterized (164,228,540); it is one of the few Trichomonas proteins that has been studied at the crystal structure level (92). Trichomonad hydrogenosomes contain components derived from complex I of the mitochondrial respiratory chain that help to maintain redox balance by reoxidizing NADH from the malic enzyme reaction (209).…”

Section: Fig 11mentioning

confidence: 99%

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Müller

Mentel

Hellemond

et al. 2012

Microbiol Mol Biol Rev

695

904

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SUMMARY Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.

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“…3A). As a control, Western analysis was performed using antisera against ferredoxin, a protein with three cysteine residues that coordinate iron (34).…”

Section: Isolation and Characterization Of Hmp35 A Hydrogenosomalmentioning

confidence: 99%

Trichomonas vaginalis Hmp35, a Putative Pore-forming Hydrogenosomal Membrane Protein, Can Form a Complex in Yeast Mitochondria

Dyall¹,

Lester

Schneider

et al. 2003

Journal of Biological Chemistry

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An abundant integral membrane protein, Hmp35, has been isolated from hydrogenosomes of Trichomonas vaginalis. This protein has no known homologue and exists as a stable 300-kDa complex, termed HMP35, in membranes of the hydrogenosome. By using blue native gel electrophoresis, we found the HMP35 complex to be stable in 2 M NaCl and up to 5 M urea. The endogenous Hmp35 protein was largely protease-resistant. The protein has a predominantly ␤-sheet structure and predicted transmembrane domains that may form a pore. Interestingly, the protein has a high number of cysteine residues, some of which are arranged in motifs that resemble the RING finger, suggesting that they could be coordinating zinc or another divalent cation. Our data show that Hmp35 forms one intramolecular but no intermolecular disulfide bonds. We have isolated the HMP35 complex by expressing a His-tagged Hmp35 protein in vivo followed by purification with nickel-agarose beads. The purified 300-kDa complex consists of mostly Hmp35 with lesser amounts of 12-, 25-27-, and 32-kDa proteins. The stoichiometry of proteins in the complex indicates that Hmp35 exists as an oligomer. Hmp35 can be targeted heterologously into yeast mitochondria, despite the lack of homology with any yeast protein, demonstrating the compatibility of mitochondrial and hydrogenosomal protein translocation machineries.Trichomonas vaginalis is a deep-branching protist that lacks archetypal eukaryotic "aerobic" organelles, specifically mitochondria and peroxisomes. This microaerophilic human-infective parasite carries out fermentative carbohydrate metabolism within hydrogenosomes. Hydrogenosomes are bounded by double membranes and produce ATP by substrate level phosphorylation (1). Hydrogenosomes are also found in certain chytrids, ciliates, and fungi, in lineages that are phylogenetically distant to the Parabasalian lineage to which trichomonads belong (1-4). Currently, several lines of evidence support a common endosymbiotic ancestry for hydrogenosomes and mitochondria, despite their distinct metabolic pathways (3,5,6). Although the origin of hydrogenosomes within ciliate and fungi lineages is debated, these lineages branch with mitochondria-containing groups and the hydrogenosomes confined therein exhibit strong similarity to ciliate and fungal mitochondria (7,8).Trichomonad hydrogenosomes, on the other hand, are markedly less similar to mitochondria. These organelles lack a genome (9) which would have provided a means to investigate their endosymbiotic origin as has been elegantly and convincingly done for mitochondria (10). In lieu of this, we and others have attempted to define the relationship between trichomonad hydrogenosomes and mitochondria by examining the origin of their chaperonins, metabolic enzymes, and membrane proteins. Chaperonin genes, specifically heat shock protein (Hsp) 1 70, cpn60,, and the IscS enzyme, involved in FeS cluster formation (15) appear to have a mitochondrial origin. However, analyses of metabolic enzymes such as hydrogenase (16,17), which is typi...

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Expression and Spectroscopic Characterization of the Hydrogenosomal [2Fe-2S] Ferredoxin from the Protozoan

Cited by 26 publications

References 29 publications

Purification and Biophysical Characterization of a New [2Fe-2S] Ferredoxin from Azotobacter vinelandii, a Putative [Fe-S] Cluster Assembly/Repair Protein

Purification and Biophysical Characterization of a New [2Fe-2S] Ferredoxin from Azotobacter vinelandii, a Putative [Fe-S] Cluster Assembly/Repair Protein

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Trichomonas vaginalis Hmp35, a Putative Pore-forming Hydrogenosomal Membrane Protein, Can Form a Complex in Yeast Mitochondria

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