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

Anurag Srivastava, M Hirasawa, M Bhalla, J S Chung, J P Allen, M K Johnson, Jatindra Tripathy, L M Rubio, B Vaccaro, S Subramanian, E Flores, Masoud Zabet Moghaddam, K Stitle, David Knaff

Research output: Contribution to journalArticlepeer-review

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 percen
Original languageEnglish
Pages (from-to)4343-4353
JournalBiochemistry
StatePublished - Jun 25 2013

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