Selective chemical modification of amino acid residues in the flavin adenine dinucleotide binding site of NADPH-ferredoxin reductase.
1. An apo-NADPH-ferredoxin reductase was prepared from holo-NADPH-ferredoxin reductase (EC 1.18.1.2) from bovine adrenocortical mitochondria. 2. Amino acid residues of the apo-reductase were modified selectively, to identify the FAD-binding site of the reductase, with chemical reagents such as diethylpyrocarbonate, 5,5'-dithiobis(2-nitrobenzoate), tetranitromethane, pyridoxal 5'-phosphate, p-nitrophenylglyoxal, diisopropylfluorophosphate and N-bromosuccinimide. The binding of FAD to the apo-reductase was measured as quenching of the fluorescence of FAD caused by the binding between apo-reductase and FAD. The quenching was blocked when the apo-reductase was modified with diethylpyrocarbonate and restored on the addition of hydroxylamine. 3. The blocking of the quenching occurred in a competitive manner as to FAD in the presence of diethylpyrocarbonate. However, when the apo-reductase was modified with 5,5'-dithiobis(2-nitrobenzoate), the blocking of the quenching occurred in a non-competitive manner. 4. These results suggested that a histidyl residue of the apo-reductase is essential for the binding of FAD to the reductase. This was confirmed by amino acid sequencing of the modified apo-reductase.
Yamazaki
M
M
Department of Biochemistry, Kagawa Medical School, Japan.
Ohnishi
T
T
Ichikawa
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; D004228:Dithionitrobenzoic Acid; D005287:Ferredoxin-NADP Reductase; D004047:Diethyl Pyrocarbonate