Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase

Anurag P. Srivastava, James P. Allen, Brian J. Vaccaro, Masakazu Hirasawa, Suzanne Alkul, Michael K. Johnson, David B. Knaff

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Abstract

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.

Original languageEnglish
Pages (from-to)5557-5568
Number of pages12
JournalBiochemistry
Volume54
Issue number36
DOIs
Publication statusPublished - Sep 15 2015

ASJC Scopus subject areas

  • Biochemistry

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    Srivastava, A. P., Allen, J. P., Vaccaro, B. J., Hirasawa, M., Alkul, S., Johnson, M. K., & Knaff, D. B. (2015). Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase. Biochemistry, 54(36), 5557-5568. https://doi.org/10.1021/acs.biochem.5b00511