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Volume 271, Number 23, Issue of June 7, 1996 pp. 13680-13683
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Studies on Protein-Protein Interaction between Copper-containing Nitrite Reductase and Pseudoazurin from Alcaligenes faecalis S-6

(Received for publication, November 20, 1995, and in revised form, March 19, 1996)

Mutsuko Kukimoto , Makoto Nishiyama , Masaru Tanokura , Elinor T. Adman ^¶ and Sueharu Horinouchi

From the Department of Biotechnology and the  Biotechnology Research Center, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan and the ^¶ Department of Biological Structure, University of Washington, Seattle, Washington 98195

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 K_m 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 K_m 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.

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