PLANT PHYSIOLOGY , Vol 114, Issue 3 1047-1053, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
The Ferredoxin-Binding Site of Ferredoxin:Nitrite Oxidoreductase (Differential Chemical Modification of the Free Enzyme and Its Complex with Ferredoxin)
M. M. Dose, M. Hirasawa, S. Kleis-SanFrancisco, E. L. Lew and D. B. Knaff
Department of Chemistry and Biochemistry (M.M.D., M.H., E.L.L., D.B.K.), and Institute for Biotechnology (S.K.-S., D.B.K.), Texas Tech University, Lubbock, Texas 79409-1061
Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite
reductase was treated with either the arginine-modifying reagent
phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5[prime]-phosphate
under conditions where only the Fd-binding affinity of the enzyme was
affected and where complex formation between Fd and the enzyme prevented
the inhibition by either reagent. Modification with [14C]phenylglyoxal
allowed the identification of two nitrite reductase arginines, R375 and
R556, that are protected by Fd against labeling. Modification of nitrite
reductase with pyridoxal-5[prime]-phosphate, followed by reduction with
NaBH4, allowed the identification of a lysine, K436, that is protected by
Fd against labeling. Positive charges are present at these positions in all
of the Fd-dependent nitrite reductases for which sequences are available,
suggesting that these amino acids are directly involved in electrostatic
binding of Fd to the enzyme.
