Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6.
Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wild-type pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.
Kukimoto
M
M
Department of Biotechnology, University of Tokyo, Japan.
Nishiyama
M
M
Ohnuki
T
T
Turley
S
S
Adman
E T
ET
Horinouchi
S
S
Beppu
T
T
D000409:Alanine; D000421:Alcaligenes / Q000201:enzymology*; D001224:Aspartic Acid; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003300:Copper / Q000032:analysis*; D018360:Crystallography, X-Ray; D004579:Electron Transport; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
C047163:pseudoazurin; D001400:Azurin; D001224:Aspartic Acid; D003300:Copper; D009572:Nitrite Reductases; D008239:Lysine; D000409:Alanine