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Investigation of the electron transport chain to and the catalytic activity of the diheme cytochrome c peroxidase CcpA of Shewanella oneidensis.

ABSTRACT: Bacterial diheme c-type cytochrome peroxidases (BCCPs) catalyze the periplasmic reduction of hydrogen peroxide to water. The gammaproteobacterium Shewanella oneidensis produces the peroxidase CcpA under a number of anaerobic conditions, including dissimilatory iron-reducing conditions. We wanted to understand the function of this protein in the organism and its putative connection to the electron transport chain to ferric iron. CcpA was isolated and tested for peroxidase activity, and its structural conformation was analyzed by X-ray crystallography. CcpA exhibited in vitro peroxidase activity and had a structure typical of diheme peroxidases. It was produced in almost equal amounts under anaerobic and microaerophilic conditions. With 50 mM ferric citrate and 50 ?M oxygen in the growth medium, CcpA expression results in a strong selective advantage for the cell, which was detected in competitive growth experiments with wild-type and ?ccpA mutant cells that lack the entire ccpA gene due to a markerless deletion. We were unable to reduce CcpA directly with CymA, MtrA, or FccA, which are known key players in the chain of electron transport to ferric iron and fumarate but identified the small monoheme ScyA as a mediator of electron transport between CymA and BCCP. To our knowledge, this is the first detailed description of a complete chain of electron transport to a periplasmic c-type cytochrome peroxidase. This study furthermore reports the possibility of establishing a specific electron transport chain using c-type cytochromes.

SUBMITTER: Schutz B

PROVIDER: S-EPMC3165401 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Investigation of the electron transport chain to and the catalytic activity of the diheme cytochrome c peroxidase CcpA of Shewanella oneidensis.

Schütz Björn B Seidel Julian J Sturm Gunnar G Einsle Oliver O Gescher Johannes J

Applied and environmental microbiology 20110708 17

Bacterial diheme c-type cytochrome peroxidases (BCCPs) catalyze the periplasmic reduction of hydrogen peroxide to water. The gammaproteobacterium Shewanella oneidensis produces the peroxidase CcpA under a number of anaerobic conditions, including dissimilatory iron-reducing conditions. We wanted to understand the function of this protein in the organism and its putative connection to the electron transport chain to ferric iron. CcpA was isolated and tested for peroxidase activity, and its struct ...[more]

PMID: 21742904

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Project description:Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design.

Project description:UnlabelledShewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the ?bfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria.ImportanceExtracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.

Dataset Information

Investigation of the electron transport chain to and the catalytic activity of the diheme cytochrome c peroxidase CcpA of Shewanella oneidensis.

Publications

Investigation of the electron transport chain to and the catalytic activity of the diheme cytochrome c peroxidase CcpA of Shewanella oneidensis.

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