NMR spectroscopic studies of two 7iron ferredoxins.
Item
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Title
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NMR spectroscopic studies of two 7iron ferredoxins.
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Identifier
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AAI9130303
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identifier
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9130303
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Creator
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Cheng, Hong.
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Contributor
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Adviser: William V. Sweeney
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Date
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1991
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Chemistry, Physical
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Abstract
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Azotobacter vinelandii ferredoxin I and Pseudomonas putida ferredoxin each contains one (3Fe-4S) cluster and one (4Fe-4S) cluster. They have homologous amino acid sequences. The structures of these two 7Fe ferredoxins are considered to be essentially the same.;The {dollar}\sp1{dollar}H NMR spectra of these two 7Fe ferredoxins look very similar. Six peaks are well resolved in the region downfield of 10 ppm. T{dollar}\sb1{dollar} relaxation time measurements, temperature dependence studies, pH titrations and deuterium labeling experiments have been performed. Peaks A-E are pH titratable and they are from {dollar}\beta{dollar}-protons on iron-bound cysteines. Peak F is pH independent, and it resolves into two resonances with approximately equal intensity at temperatures below 15{dollar}\sp\circ{dollar}C or above 25{dollar}\sp\circ{dollar}C. Neither of the resolved resonances arises from an {dollar}\alpha{dollar}- or {dollar}\beta{dollar}-cysteinyl proton. In addition, a partial assignment of the ring system protons of the two 7Fe ferredoxins has been made.;Nuclear Overhauser effects are observed between paramagnetically shifted resonances. 1D steady state NOE experiments show that peaks A and B are from a geminal pair of {dollar}\beta{dollar}-protons. These single proton resonances arise from a cysteine bound to the 3Fe center. Similarly, it is shown that peak C is from one of the {dollar}\beta{dollar}-protons of Cys16 and peak E is from one of the {dollar}\beta{dollar}-protons of Cys45. Other cysteinyl protons are correlated to peaks A-E based on the results from a NOESY experiment with a short mixing time of 5 ms.;P. putida ferredoxin was {dollar}\sp{13}{dollar}C enriched at the {dollar}\beta{dollar}-cysteinyl carbon atoms through direct incorporation of labeled cysteine. Eight peaks, 1-8, are observed in the {dollar}\sp{13}{dollar}C NMR spectrum of this {dollar}\sp{13}{dollar}C enriched ferredoxin. The correlation between {dollar}\beta{dollar}-cysteinyl carbon and proton resonances have been studied by heteronuclear correlated experiments. The chemical shifts and temperature dependencies of carbon peaks 7 and 8 and their correlated proton resonances indicate that peaks 7 and 8 are from two cysteines with free sulfhydryl groups. Thus, only seven cysteines in these two 7Fe ferredoxins are iron-bound. This result supports the view that the correct structure of the 3Fe center is Fe{dollar}\sb3{dollar}S{dollar}\sb4{dollar}(S{dollar}\sp{lcub}\rm Cys{rcub})\sb3{dollar}, as reported in the revised X-ray structure of Azotobacter vinelandii ferredoxin I.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
