Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus,
Pauleta, S. R., Lu Y., Goodhew C. F., Moura I., Pettigrew G. W., and Shelnutt J. A.
, Biochemistry, Jun 5, Volume 40, Number 22, p.6570-6579, (2001)
AbstractThe structural changes in the heme macrocycle and substituents caused by binding of Ca2+ to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca2+-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca2+ or Mg2+. This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca2+ binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca2+ binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.
A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase,
Pauleta, S. R., Cooper A., Nutley M., Errington N., Harding S., Guerlesquin F., Goodhew C. F., Moura I., Moura J. J., and Pettigrew G. W.
, Biochemistry, Nov 23, Volume 43, Number 46, p.14566-76, (2004)
AbstractPseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.
Characterization of representative enzymes from a sulfate reducing bacterium implicated in the corrosion of steel,
Pereira, A. S., Franco R., Feio M. J., Pinto C., Lampreia J., Reis M. A., Calvete J., Moura I., Beech I., Lino A. R., and Moura J. J.
, Biochem Biophys Res Commun, Apr 16, Volume 221, Number 2, p.414-21, (1996)
AbstractThis communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed.
Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135 - Evidence for modulation of the rate of intramolecular electron transfer through nitrite binding to the type 2 copper center,
Pinho, D., Besson S., Brondino C. D., de Castro B., and Moura I.
, European Journal of Biochemistry, Jun, Volume 271, Number 12, p.2361-2369, (2004)
AbstractThe nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO2-. Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme.