A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.
Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.
Hirasawa
M
M
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061, USA.
Dose
M M
MM
Kleis-SanFrancisco
S
S
Hurley
J K
JK
Tollin
G
G
Knaff
D B
DB
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D005288:Ferredoxins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea; D014364:Tryptophan / Q000378:metabolism*
D005288:Ferredoxins; D014364:Tryptophan; D009572:Nitrite Reductases; C023099:ferredoxin-nitrite oxidoreductase