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The structure of the Met144Leu mutant of copper nitrite reductase from Alcaligenes xylosoxidans provides the first glimpse of a protein–protein complex with azurin II

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Abstract

Cu-containing nitrite reductases (NiRs) perform the reduction of nitrite to NO via an ordered mechanism in which the delivery of a proton and an electron to the catalytic type 2 Cu site is highly orchestrated. Electron transfer from a redox partner protein, azurin or pseudoazurin, to the type 1 Cu site is assumed to occur through the formation of a protein–protein complex. We report here a new crystal form in space group P212121 of the Met144Leu mutant of NiR from Alcaligenes xylosoxidans (AxNiR), revealing a head-to-head packing motif involving residues around the hydrophobic patch of domain 1. Superposition of the structure of azurin II with that of domain 1 of one of the Met144Leu molecules provides the first glimpse of an azurin II–NiR protein–protein complex. Mutations of two of the residues of AxNiR, Trp138His (Barrett et al. in Biochemistry 43:16311–16319, 2004) and Met87Leu, highlighted in the AxNiR–azurin complex, results in substantially decreased activity when azurin is used as the electron donor instead of methyl viologen, providing direct evidence for the importance of this region for complex formation.

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Notes

  1. The numbering is that of NiR from Alcaligenes xylosoxidans (AxNiR).

  2. A preliminary report [17] for a complex between pseudoazurin and Achromobacter cycloclastes NiR was given at a conference and in its proceedings. However, no further report or a Protein Data Bank (PDB) deposition has emerged.

  3. We previously determined the crystal structure of Met144Leu AxNiR in space group H3, PDB code 2bp0 [4].

  4. The Met87Leu mutant was produced using a similar protocol to that used for the Met144Leu mutant [4]. NiR activity was determined using two independent methods using methyl viologen/dithionite ion mixed assay or azurin as a reductant as described in [6, 37].

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Acknowledgements

The authors would like to thank Roger Harris (John Innes Centre) and members of the Daresbury Molecular Biophysics Group for their help and interest. We also acknowledge Mark Ellis (Daresbury) for assistance with the activity measurement for the M87L mutant of AxNiR. We would also like to thank both STFC and BBSRC for the provision of facilities at Daresbury Laboratory and the John Innes Centre, respectively. This work was supported by the BBSRC’s Biomolecular Sciences programme (grant numbers 719/B14224 and BBD0162901).

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Authors and Affiliations

  1. Molecular Biophysics Group, STFC Daresbury Laboratory, Warrington, WA4 4AD, UK

    Konstantinos Paraskevopoulos, Michael A. Hough, Robert R. Eady & S. Samar Hasnain

  2. School of Biomolecular Sciences, Liverpool John Moores University, Liverpool, L3 5AF, UK

    Konstantinos Paraskevopoulos & S. Samar Hasnain

  3. Max-Planck-Institut für Terrestrische Mikrobiologie, 35043, Marburg, Germany

    R. Gary Sawers

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  1. Konstantinos Paraskevopoulos
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  2. Michael A. Hough
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  3. R. Gary Sawers
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  4. Robert R. Eady
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  5. S. Samar Hasnain
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Correspondence to S. Samar Hasnain.

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Paraskevopoulos, K., Hough, M.A., Sawers, R.G. et al. The structure of the Met144Leu mutant of copper nitrite reductase from Alcaligenes xylosoxidans provides the first glimpse of a protein–protein complex with azurin II. J Biol Inorg Chem 12, 789–796 (2007). https://doi.org/10.1007/s00775-007-0233-y

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