Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain

Sousa, Patrícia M. Paes de; Rodrigues, David; Timóteo, Cristina G.; Gonçalves, Maria Eduarda; Pettigrew, Graham W.; Moura, I.; Moura, José J. G.; Santos, Margarida M. Correia dos

doi:10.1007/s00775-011-0785-8

Cited by 5 publications

(2 citation statements)

References 34 publications

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“…Electrocatalytic potentials of bacterial CcP enzymes as a function of substrate concentration, as depicted as in ref . The recombinant Ne enzyme (solid squares) is compared to Ne F81W (open squares), activated So enzyme (solid triangles), So F102W (open triangles), as well as Geobacter CcP (circles, taken from ref ), and unactivated Pseudomonas enzyme (diamonds, taken from ref ).…”

Section: Resultsmentioning

confidence: 99%

“…Films generated using oxidized (“closed”) enzyme could not be converted to the open state by poising at highly oxidizing potentials (>700 mV vs SHE). Previous observations have suggested that the highly polar graphite surface can select for certain conformations of enzymes or promote unfolding of His/Met cytochromes. − In the case of So CcP, the “closed” to “open” conformational shift presumably results in the motion of surface loops rich in both positively and negatively charged amino acid side chains. It is likely that these differing charge distributions account for different affinities to the graphite surface.…”

Section: Discussionmentioning

confidence: 99%

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Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

2015

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The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

2015

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Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production

Roger

Poulpiquet²,

Ciaccafava³

et al. 2013

Anal Bioanal Chem

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How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein-protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.

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Reduction of hydrogen peroxide in gram-negative bacteria – bacterial peroxidases

Nóbrega

Pauleta

2019

Advances in Microbial Physiology

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Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain

Cited by 5 publications

References 34 publications

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production

Reduction of hydrogen peroxide in gram-negative bacteria – bacterial peroxidases

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