Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase

Anurag P. Srivastava, Emily P. Hardy, James Paul Allen, Brian J. Vaccaro, Michael K. Johnson, David B. Knaff

Research output: Contribution to journalArticle

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Abstract

An in silico model for the 1:1 ferredoxin (Fd)/nitrate reductase (NR) complex, using the known structure of Synechocystis sp. PCC 6803 Fd and the in silico model of Synechococcus sp. PCC 7942 NR, is used to map the interaction sites that define the interface between Fd and NR. To test the electrostatic interactions predicted by the model complex, five positively charged NR amino acids (Arg43, Arg46, Arg197, Lys201, and Lys614) and a negatively charged amino acid (Glu219) were altered using site-directed mutagenesis and characterized by activity measurements, metal analysis, and electron paramagnetic resonance (EPR) studies. All of the charge replacement variants retained wild-type levels of activity with reduced methyl viologen (MV), but a significant decrease in activity was observed for the R43Q, R46Q, K201Q, and K614Q variants when reduced Fd served as the electron donor. EPR analysis as well as the Fe and Mo analyses showed that loss of activity observed with these variants was not the consequence of perturbation of the Mo center or [4Fe-4S] cluster. Therefore, the loss of the Fd-linked specific activity observed with these variants can be explained only by invoking a role for Arg43, Arg46, Lys201, and Lys614 in Fd binding. The R43Q, R46Q, K201Q, and K614Q NR variants also showed a decreased binding affinity for Fd, compared to that of wild-type NR, supporting a key role of these four positively charged residues in the productive binding of Fd.

Original languageEnglish
Pages (from-to)5582-5592
Number of pages11
JournalBiochemistry
Volume56
Issue number41
DOIs
Publication statusPublished - Oct 17 2017

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Nitrate Reductase
Ferredoxins
Binding Sites
Electron Spin Resonance Spectroscopy
Computer Simulation
Paramagnetic resonance
Metal analysis
Synechococcus
Synechocystis
Amino Acids
Mutagenesis
Paraquat
Site-Directed Mutagenesis
Coulomb interactions
Static Electricity
Metals
Electrons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase. / Srivastava, Anurag P.; Hardy, Emily P.; Allen, James Paul; Vaccaro, Brian J.; Johnson, Michael K.; Knaff, David B.

In: Biochemistry, Vol. 56, No. 41, 17.10.2017, p. 5582-5592.

Research output: Contribution to journalArticle

Srivastava, Anurag P. ; Hardy, Emily P. ; Allen, James Paul ; Vaccaro, Brian J. ; Johnson, Michael K. ; Knaff, David B. / Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase. In: Biochemistry. 2017 ; Vol. 56, No. 41. pp. 5582-5592.
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