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Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: Characterization of an active site isoleucine

¹ Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada
² Department of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia, Canada

Reprint requests to: Michael E. Murphy, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada; e-mail: memurphy{at}interchange.ubc.ca; fax: (604) 822-6041.

Unlike the heme cd₁-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 Å) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd₁ NiRs.

(The atomic coordinates for I257V[NO₂^-], I257L[NO₂^-], I257A[NO₂^-], I257T[NO₂^-], I257M[NO₂^-] and I257G[NO₂^-] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)

Abbreviations: NiR, nitrite reductase • CuNiR, copper-containing nitrite reductase • AfNiR, NiR from Alcaligenes faecalis S-6 • AcNiR, NiR from Achromobacter cycloclastes • [NO₂^-], nitrite-soaked crystal structure • FT-IR, Fourier transform infrared spectroscopy • EPR, electron paramagnetic resonance • EXAFS, extended X-ray fine absorption structure spectroscopy

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