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, Morgan 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 2013

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