Characterization of the Type III sulfide:quinone oxidoreductase from Caldivirga maquilingensis and its membrane binding.
ABSTRACT Sulfide:quinone oxidoreductases (SQRs) are ubiquitous enzymes which have multiple roles: sulfide detoxification, energy generation by providing electrons to respiratory or photosynthetic electron transfer chains, and sulfide homeostasis. A recent structure-based classification defines 6 groups of putative SQRs (I - VI), and representatives of all but group III have been confirmed to have sulfide oxidase activity. In the current work, we report the first characterization of a predicted group III SQR from Caldivirga maquilingensis, and confirm that this protein is a sulfide oxidase. The gene encoding the enzyme was cloned, and the protein was expressed in E. coli and purified. The enzyme oxidizes sulfide using decylubiquinone as an electron acceptor, and is inhibited by aurachin C and iodoacetamide. Analysis of the amino acid sequence indicates that the C. maquilingensis SQR has two amphiphilic helices at the C-terminus but lacks any transmembrane helices. This suggests that C. maquilingensis SQR interacts with the membrane surface and that the interactions are mediated by the C-terminal amphiphilic helices. Mutations within the last C-terminal amphiphilic helix resulted in a water-soluble form of the enzyme which, remarkably, retains full SQR activity using decylubiquinone as the electron acceptor. Mutations at one position, L379, also located in the C-terminal amphiphilic helix, inactivated the enzyme by preventing the interaction with decylubiquinone. It is concluded that the C-terminal amphiphilic helix is important for membrane binding and for forming part of the pathway providing access of the quinone substrate to the protein-bound flavin at the enzyme active site.
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ABSTRACT: Abstract Type 2 NADH dehydrogenase (NDH-2) is a single-subunit membrane-associated flavoenzyme that is part of the respiratory chain of many prokaryotes. The enzyme catalyzes the electron transfer from NADH to quinone but is not directly coupled to the generation of a proton motive force. The purpose of the current work is to compare two different NDH-2s that are encoded in strains of Thermus thermophilus. The aerobic T. thermophilus HB27 strain expresses one NDH-2 that has been previously isolated and characterized. In this work it is shown that a gene, which is misannotated as an NADH oxidase, encodes this enzyme. Unlike HB27, strain NAR1 of T. thermophilus is capable of partial denitrification, and its genome contains, in addition, the ncrN gene that encodes a second putative NDH-2. Of particular interest is that ncrN is part of an operon (nrcDEFN) that is proposed to encode a protein complex specifically required for nitrate reduction. In this work, the nrcN gene has the activity expected of an NDH-2, and functions independently of other components of the putative Nrc complex. The biochemical properties of the two NDH-2 enzymes are compared. Efforts to demonstrate that NrcN is part of a multiprotein complex were not successful. However, the NrcE protein was expressed in E. coli and shown to be a membrane-bound protein containing heme B.Biological Chemistry 01/2013; · 2.69 Impact Factor
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ABSTRACT: Snottites are extremely acidic (pH 0–2) biofilms that form on the walls and ceilings of hydrogen sulfide-rich caves. Recent work suggests that microbial communities including snottites and related cave wall biofilms accelerate cave formation by oxidizing sulfide to sulfuric acid. Therefore, we used full-cycle rRNA methods and metagenomics to explore the community composition and sulfur metabolism of snottites from the sulfidic Frasassi and Acquasanta cave systems, Italy. Acquasanta snottites were dominated by strains of Acidithiobacillus thiooxidans, with a smaller population of Ferroplasma sp. Frasassi snottites were also dominated by At. thiooxidans but with a more diverse community including relatives of ‘G-plasma’ (Thermoplasmatales), Acidimicrobium, and rare taxa. We identified diverse homologues of sulfide:quinone oxidoreductase (SQR) in the metagenomic datasets. Based on phylogenetic analysis, the numerically dominant At. thiooxidans populations have four different types of SQR, while Ferroplasma has two and Acidimicrobium and G-plasma each have one. No other genetic evidence for sulfur oxidation was detected for either Acidimicrobium or G-plasma, suggesting that they do not generate sulfuric acid. Our results confirm earlier findings that At. thiooxidans is the dominant primary producer and sulfide oxidizer in sulfidic cave snottites.Geomicrobiology 03/2014; 31(3). · 1.80 Impact Factor