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
Hauptverfasser: Carrillo, N, Arana, J L, Vallejos, R H
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container_title The Journal of biological chemistry
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creator Carrillo, N
Arana, J L
Vallejos, R H
description 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.
doi_str_mv 10.1016/S0021-9258(19)69066-6
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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.</description><subject>Binding Sites</subject><subject>Chloroplasts - enzymology</subject><subject>Dihydrolipoamide Dehydrogenase - metabolism</subject><subject>Ferredoxin-NADP Reductase - metabolism</subject><subject>Kinetics</subject><subject>Mathematics</subject><subject>NADH, NADPH Oxidoreductases - metabolism</subject><subject>NADP</subject><subject>Oxidation-Reduction</subject><subject>Plants - enzymology</subject><subject>Protein Binding</subject><subject>Spectrophotometry</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1981</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kG9r2zAQxsVYadNsHyEgKIyV4layrH8vQ9qthbIWtsHeFCFL50TFsTLJZsm3n5uE3ps77nnujvshNKPkmhIqbn4SUtJCl1x9pfpSaCJEIT6gCSWKFYzTPx_R5N1yhs5zfiVjVJqeolOhdMW1mqCXeYchZ-j6YFvsbKrjdtfiZYrDBtse9yvA3eBaiH3wgOvQ-dAtcQ494NjgBlICH7ehK37Mb5-v8Fj6OLYG19sMn9BJY9sMn495in5_u_u1uC8en74_LOaPhWNS90Ula2gs11JRZRXRzlWktqqRrpRcEUcrURHPSNMwK2taWZCeKVpzb8dnfcWm6Mth7ybFvwPk3qxDdtC2toM4ZCO5UIQSORr5wehSzDlBYzYprG3aGUrMG1azx2remBmqzR6rEePc7HhgqNfg36eOHEf94qCvwnL1LyQwdYhuBWtTcmEoG40lY_8BWyN_ZA</recordid><startdate>19810710</startdate><enddate>19810710</enddate><creator>Carrillo, N</creator><creator>Arana, J L</creator><creator>Vallejos, R H</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19810710</creationdate><title>An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase</title><author>Carrillo, N ; Arana, J L ; Vallejos, R H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c379t-47befa597818a809cc40ba8f7c27580c14640d30ff3a7b14ae7d381b5da083d43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1981</creationdate><topic>Binding Sites</topic><topic>Chloroplasts - enzymology</topic><topic>Dihydrolipoamide Dehydrogenase - metabolism</topic><topic>Ferredoxin-NADP Reductase - metabolism</topic><topic>Kinetics</topic><topic>Mathematics</topic><topic>NADH, NADPH Oxidoreductases - metabolism</topic><topic>NADP</topic><topic>Oxidation-Reduction</topic><topic>Plants - enzymology</topic><topic>Protein Binding</topic><topic>Spectrophotometry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Carrillo, N</creatorcontrib><creatorcontrib>Arana, J L</creatorcontrib><creatorcontrib>Vallejos, R H</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Carrillo, N</au><au>Arana, J L</au><au>Vallejos, R H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1981-07-10</date><risdate>1981</risdate><volume>256</volume><issue>13</issue><spage>6823</spage><epage>6828</epage><pages>6823-6828</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. 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subjects Binding Sites
Chloroplasts - enzymology
Dihydrolipoamide Dehydrogenase - metabolism
Ferredoxin-NADP Reductase - metabolism
Kinetics
Mathematics
NADH, NADPH Oxidoreductases - metabolism
NADP
Oxidation-Reduction
Plants - enzymology
Protein Binding
Spectrophotometry
title An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase
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