Roles of four conserved basic amino acids in a ferredoxin-dependent cyanobacterial nitrate reductase

Anurag P. Srivastava, Masakazu Hirasawa, Megha Bhalla, Jung Sung Chung, James P. Allen, Michael K. Johnson, Jatindra N. Tripathy, Luis M. Rubio, Brian Vaccaro, Sowmya Subramanian, Enrique Flores, Masoud Zabet-Moghaddam, Kyle Stitle, David B. Knaff

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Abstract

The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.

Original languageEnglish
Pages (from-to)4343-4353
Number of pages11
JournalBiochemistry
Volume52
Issue number25
DOIs
Publication statusPublished - Jun 25 2013

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Srivastava, A. P., Hirasawa, M., Bhalla, M., Chung, J. S., Allen, J. P., Johnson, M. K., Tripathy, J. N., Rubio, L. M., Vaccaro, B., Subramanian, S., Flores, E., Zabet-Moghaddam, M., Stitle, K., & Knaff, D. B. (2013). Roles of four conserved basic amino acids in a ferredoxin-dependent cyanobacterial nitrate reductase. Biochemistry, 52(25), 4343-4353. https://doi.org/10.1021/bi400354n