Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe-2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the [2Fe-2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements.
Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties / A. Aliverti, W.R. Hagen, G. Zanetti. - In: FEBS LETTERS. - ISSN 0014-5793. - 368:2(1995 Jul 17), pp. 220-224.
Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties
A. AlivertiPrimo
;G. ZanettiUltimo
1995
Abstract
Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe-2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the [2Fe-2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements.Pubblicazioni consigliate
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