Studies on protein-protein interaction between copper-containing nitrite reductase and pseudoazurin from Alcaligenes faecalis S-6.
Site-directed mutagenesis of a copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to identify the amino acid residues involved in interaction with its redox partner, pseudoazurin, in which four positively charged residues were previously shown to be important in the interaction. Ten negatively charged residues located on the surface of NIR were replaced independently by alanine or serine. All the altered NIRs showed CD spectra and optical spectra identical to those of wild-type NIR, suggesting that all the replacements caused no gross change in the overall structure or in the environment of type 1 copper site. Kinetic analysis of electron transfer between pseudoazurin and altered NIRs revealed that the replacement of Glu-118, Glu-197, Asp-201, Glu-204, or Asp-205 by Ala caused a significant increase in the Km value for pseudoazurin compared with that of wild-type NIR. Furthermore, the simultaneous replacement of three of these residues (Glu-118, Glu-197, and Asp-201) caused a further increase in the Km value. These results suggested that the negatively charged residues are involved in electrostatic interaction with pseudoazurin. Kinetic analyses of the altered NIRs (E118A, E197A, or D201A) with altered pseudoazurins (K10A, K57A, or K77A) implicate specific pairs of the charged residues that are involved in electrostatic interaction between NIR and pseudoazurin.
Kukimoto
M
M
Department of Biotechnology and the Biotechnology Research Center, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan.
Nishiyama
M
M
Tanokura
M
M
Adman
E T
ET
Horinouchi
S
S
D000421:Alcaligenes / Q000235:genetics / Q000378:metabolism*; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003300:Copper / Q000737:chemistry; D004563:Electrochemistry; D004579:Electron Transport; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation
D009838:Oligodeoxyribonucleotides; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; D009565:Nitrate Reductases; D050901:Nitrate Reductase