Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase.
The 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.
Pauleta
Sofia R
SR
ReQuimte, Centro de Química Física e Biotecnologia, FCT/UNL, Quinta da Torre, 2829-516 Caparica, Portugal.
Guerlesquin
Françoise
F
Goodhew
Celia F
CF
Devreese
Bart
B
Van Beeumen
Jozef
J
Pereira
Alice S
AS
Moura
Isabel
I
Pettigrew
Graham W
GW
D001400:Azurin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004579:Electron Transport; D015964:Gene Expression Regulation, Bacterial; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D042084:Paracoccus pantotrophus / Q000201:enzymology*; D010449:Peptide Mapping; D011485:Protein Binding; D055672:Static Electricity; D013379:Substrate Specificity
D008667:Metalloproteins; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; D003578:Cytochrome-c Peroxidase; D008239:Lysine