Roles of Four Conserved Basic Amino Acids in a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase

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• 作者：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
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：June 25, 2013
• 年：2013
• 卷：52
• 期：25
• 页码：4343-4353
• 全文大小：460K
• 年卷期：v.52,no.25(June 25, 2013)
• ISSN：1520-4995

文摘

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鈥檚 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.