Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties

FEBS Letters

Aliverti, Alessandro¹  Hagen, Wilfred R.²  Zanetti, Giuliana¹ 

[1] Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Milan, Italy;Department of Biochemistry, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands

关键词: Ferredoxin I;    Iron-sulfur cluster;    Site-directed mutagenesis;    Redox potential;    Cyclic voltammetry;    EPR;    FNR;    ferredoxin-NADP+ reductase;    FdI;    ferredoxin I;    Fd-FNR;    fusion between FdI and FNR;    NHE;    normal hydrogen electrode;   

DOI  :  10.1016/0014-5793(95)00648-S

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown