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Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus
- De Smet, Lina, Pettigrew, Graham W., Van Beeumen, Jozef J.
- European journal of biochemistry 2001 v.268 no.24 pp. 6559-6568
- Escherichia coli, Rhodobacter capsulatus, calcium, catalytic activity, cytochrome c, cytochrome-c peroxidase, enzymatic treatment, ethylene glycol tetraacetic acid, horses, hydrogen peroxide, photosynthetic bacteria, physicochemical properties, recombinant proteins, signal peptide, structural genes, titration
- The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone overâexpressing the BCCP structural gene. BCCP from Rb.âcapsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb.âcapsulatus was cloned through the construction of a 7âkb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21âaminoâacid Nâterminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physicoâchemical properties as the native enzyme. Spectrophotometric titration established the presence of a highâpotential (Em=+270âmV) and a lowâpotential haem (between â190âmV and â310âmV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a highâspin state at the lowâpotential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities (âturnover numberâ) are 85â800Â·minâ1 and 63â600Â·minâ1, respectively. These activities are the highest ever found for a BCCP.