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How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

ABSTRACT: Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E'0 = -231 mV) as compared with that of TlpA (E'0 = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10(4) M(-1) s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu(A)-CoxB complex may be bypassed in vivo by high environmental Cu(2+) concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.

SUBMITTER: Abicht HK

PROVIDER: S-EPMC4239598 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

Abicht Helge K HK Schärer Martin A MA Quade Nick N Ledermann Raphael R Mohorko Elisabeth E Capitani Guido G Hennecke Hauke H Glockshuber Rudi R

The Journal of biological chemistry 20141001 47

Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfe ...[more]

PMID: 25274631

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Project description:UnlabelledCopper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb3-type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb3-Cox production in bacteria like R. capsulatus.ImportanceCopper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochrome c oxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochrome c oxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochrome c oxidase in the cell.

Dataset Information

How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

Publications

How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

Similar Datasets

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