Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6.
Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Akaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.
Kukimoto
M
M
Department of Biotechnology, The University of Tokyo, Japan.
Nishiyama
M
M
Tanokura
M
M
Murphy
M E
ME
Adman
E T
ET
Horinouchi
S
S
D000421:Alcaligenes / Q000201:enzymology*; D001400:Azurin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000032:analysis; D018360:Crystallography, X-Ray; D004579:Electron Transport; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; C050107:cupredoxin; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; D009572:Nitrite Reductases