CcpA from Geobacter sulfurreducens Is a Basic Di-Heme Cytochrome c Peroxidase

Hoffmann, M.; Seidel, Julian; Einsle, Oliver

doi:10.1016/j.jmb.2009.09.001

Cited by 36 publications

(64 citation statements)

References 51 publications

(68 reference statements)

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“…CcpA His showed a maximum reaction rate of 20.2 Ϯ 0.8 mol H 2 O 2 min Ϫ1 mg Ϫ1 protein and a half-maximal rate at an H 2 O 2 concentration of 11.3 Ϯ 0.6 M. The turnover number was determined to be 23.1 s Ϫ1 . The measured reaction rates agree well with rates determined for other c-type cytochrome peroxidases (22,50).…”

Section: Resultssupporting

confidence: 82%

“…The protein did not crystallize in the as-isolated (oxidized) state but yielded well-diffracting crystals in the all-ferrous form after reduction with sodium dithionite. CcpA His forms a homodimer that is highly similar to the dimers observed in other bacterial diheme peroxidases from Pseudomonas aeruginosa (17), Nitrosomonas europaea (48), Paracoccus denitrificans (13), Marinobacter hydrocarbonoclasticus (previously Pseudomonas nautica) (11), Paracoccus pantotrophus (12), and G. sulfurreducens (22) (Fig. 2; see Fig.…”

Section: Resultssupporting

confidence: 66%

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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

Seidel

Sturm

et al. 2011

Appl Environ Microbiol

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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.

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Section: Resultssupporting

confidence: 82%

Section: Resultssupporting

confidence: 66%

Investigation of the Electron Transport Chain to and the Catalytic Activity of the Diheme Cytochrome c Peroxidase CcpA of Shewanella oneidensis

Schütz

Seidel

Sturm

et al. 2011

Appl Environ Microbiol

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“…Whereas the double mutant is constitutively active in solution (15), and the crystal structure of the enzyme revealed that the diferric form of the enzyme displays an open conformation of the active site (Figure 1B), the electrocatalysis displayed in Figure 3 is not akin to that found for the either sub-classes of C c P, showing two distinct phases of the reduction of substrate. For S134P/V135K lower potential electrocatalytic feature ( E cat,1 ) remains close to values of potential observed for the wild-type enzyme, but the additional, higher-potential feature ( E cat,2 ) is found to have E cat = +200 mV vs SHE at pH 7.…”

Section: Discussionmentioning

confidence: 99%

“…For the complete expression, mutagenesis, and purification conditions please refer to reference (15). …”

Section: Methodsmentioning

confidence: 99%

“…In an attempt to mimic the properties of the constitutively active Ne enzyme, mutants were created based on the protein sequence of the Ne C c P. The most notable construct was the S134P/V135K ( Gs numbering) double mutant, where both mutations are installed on the second loop region of Gs CcpA. This mutation allows for the enzyme to be constitutively active without the requirement for pre-reduction, and a comparison of the active site crystal structures of the wild-type and double mutant enzymes (Figure 1B) emphasizes the conformational change around the L -heme, as well as the changes in the global protein fold (Figure 1C) that allows for activity without pre-reduction of this enzyme (15). …”

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confidence: 99%

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Geobacter sulfurreducens Cytochrome c Peroxidases: Electrochemical Classification of Catalytic Mechanisms

et al. 2011

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Bacterial cytochrome c peroxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (called L, for “low reduction potential”) active site heme, and a secondary heme (H, for “high reduction potential”) associated with electron transfer, and an enzymatic activity that exists only when the H-heme is pre-reduced to the FeII oxidation state. The pre-reduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an “open” conformation allowing hydrogen peroxide to bind to the FeIII of the L-heme. Notably, the enzyme from Nitrosomonas europaea does not require pre-reduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool in distinguishing the electrocatalytic mechanisms of the Nitromonas-type of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme from Geobacter sulfurreducens, and the Geobacter S134P/V135K double mutant, which has been shown to be similar to the canonical sub-class of peroxidases and the Nitrosomonas sub-class of enzymes, respectively. Here we find that the wild-type Geobacter CcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH, yet is at a potential that is very similar to the H-heme. These findings are interpreted in terms of a model where rate-limiting intra-protein electron transfer governs the catalytic performance of the S134P/V135K enzyme.

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Phylogenetic analysis, molecular modeling, substrate–inhibitor specificity, and active site comparison of bacterial, fungal, and plant heme peroxidases

Singh

Pandey

Naaz

et al. 2012

Biotech and App Biochem

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Phylogenetic analysis of 40 heme peroxidases, belonging to both prokaryotes and eukaryotes, revealed their clustering into three major classes. Class I represented sequences from plants, bacteria, fungi, and algae, whereas classes II and III exclusively represented plant and fungal peroxidases, respectively. Modeling of three representative classes of peroxidases, belonging to each of bacterial, plant, and fungal categories, revealed a similar kind of folding; however, superimposition analysis revealed relatively more closeness between plant and fungal peroxidases than that of the bacterial peroxidase. The docking analysis of three representative modeled peroxidases with three common substrates, namely, H₂O₂, guaiacol, and ascorbate, and three arginine-specific inhibitors, namely, phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione, revealed that all three inhibitors competed for guaiacol- and ascorbate-binding sites of peroxidases, except for phenylglyoxal binding in the case of plant peroxidase. Phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione were found to be most potent inhibitors of bacterial, fungal, and plant peroxidases, respectively.

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CcpA from Geobacter sulfurreducens Is a Basic Di-Heme Cytochrome c Peroxidase

Cited by 36 publications

References 51 publications

Investigation of the Electron Transport Chain to and the Catalytic Activity of the Diheme Cytochrome c Peroxidase CcpA of Shewanella oneidensis

Investigation of the Electron Transport Chain to and the Catalytic Activity of the Diheme Cytochrome c Peroxidase CcpA of Shewanella oneidensis

Geobacter sulfurreducens Cytochrome c Peroxidases: Electrochemical Classification of Catalytic Mechanisms

Phylogenetic analysis, molecular modeling, substrate–inhibitor specificity, and active site comparison of bacterial, fungal, and plant heme peroxidases

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