An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase
Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent Kd for protection afforded by NADP+ d...
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Veröffentlicht in: | The Journal of biological chemistry 1981-07, Vol.256 (13), p.6823-6828 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+
reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent
Kd for protection afforded by NADP+ depended on the ionic strength of the medium. Nucleophylic displacement of reagent bound
to the soluble enzyme by [14C]glycine ethyl ester showed that 5 to 6 carboxyl groups/flavin were modified when the diaphorase
activity was completely inhibited. In differential labeling experiments using NADP+ as protective agent, it was shown that
enzyme inactivation was due to blocking of only 1 carboxyl group/mol. Derivatized reductase did not bind pyridine nucleotides.
Protection by NADP+ of the membrane-bound reductase was higher, and the apparent Kd for NADP+ lower, in the light than in
the dark. Inactivation increased abruptly with the external pH, indicating a progressive exposure of the carboxyl group as
the pH was raised. The results presented suggest (a) the existence of a light-driven conformational change and a pH-dependent
transition in membrane-bound ferredoxin-NADP+ reductase; (b) the presence of an essential carboxyl residue in the nucleotide
binding site of the reductase. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(19)69066-6 |