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The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach

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Abstract

A system has been developed for expressing a His-tagged form of the ferredoxin-dependent nitrite reductase of spinach in Escherichia coli. The catalytic and spectral properties of the His-tagged, recombinant enzyme are similar, but not identical, to those previously observed for nitrite reductase isolated directly from spinach leaf. A detailed comparison of the spectral, catalytic and fluorescence properties of nitrite reductase variants, in which each of the enzyme’s eight tryptophan residues has been replaced using site-directed mutagenesis by either aromatic or non-aromatic amino acids, has been used to examine possible roles for tryptophan residues in the reduction of nitrite to ammonia catalyzed by the enzyme.

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Abbreviations

CD:

Circular dichroism

EPR:

Electron paramagnetic resonance

IPTG:

Isopropyl-ß-d-thiogalactopyranoside

LB:

Luria-Bertani

MALDI-TOF:

Matrix-assisted laser desorption ionization-time of flight

NBS:

N-bromosuccinimide

SDS-PAGE:

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

WT:

Wild-type

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Acknowledgments

The authors would like to thank Prof. Steven Rothstein (University of Guelph) for a generous gift of a spinach nitrite reductase clone, Prof. Toshiharu Hase (University of Osaka) for a generous gift of a clone of the E. coli cysG gene and for many helpful discussions. This research was supported by a grant (to DBK) from the U.S. Department of Energy (DE-FG02-99ER20346) and a grant (to JFA) from the U.S. Department of Agriculture (2003-02149).

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Correspondence to David B. Knaff.

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Tripathy, J.N., Hirasawa, M., Kim, SK. et al. The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach. Photosynth Res 94, 1–12 (2007). https://doi.org/10.1007/s11120-007-9198-5

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  • DOI: https://doi.org/10.1007/s11120-007-9198-5

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