Structural basis for the mechanism of Ca2+ activation of the di-heme cytochrome c peroxidase from Pseudomonas nautica 617,
Dias, J. M., Alves T., Bonifacio C., Pereira A. S., Trincao J., Bourgeois D., Moura I., and Romão M. J.
, Structure, Jul, Volume {12}, Number {6}, 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA, p.{961-973}, (2004)
AbstractCytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.
Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase,
Pauleta, S. R., Guerlesquin F., Goodhew C. F., Devreese B., Van Beeumen J., Pereira A. S., Moura I., and Pettigrew G. W.
, Biochemistry, Volume {43}, Number {35}, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, p.{11214-11225}, (2004)
AbstractThe gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.