Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase.
The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.
van de Kamp
M
M
Chemistry Department, Gorlaeus Laboratories, Leiden University, The Netherlands.
Silvestrini
M C
MC
Brunori
M
M
Van Beeumen
J
J
Hali
F C
FC
Canters
G W
GW
D000595:Amino Acid Sequence; D001400:Azurin / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D003574:Cytochrome c Group / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004579:Electron Transport; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa; D012995:Solubility; D013329:Structure-Activity Relationship; D013696:Temperature
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D001400:Azurin; C017167:cytochrome C(551); D009572:Nitrite Reductases