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Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties
Author(s) -
Aliverti Alessandro,
Hagen Wilfred R.,
Zanetti Giuliana
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00648-s
Subject(s) - ferredoxin , ferredoxin thioredoxin reductase , chemistry , spinach , iron–sulfur cluster , biochemistry , reductase , cyclic voltammetry , electron paramagnetic resonance , ferredoxin—nadp(+) reductase , enzyme , thioredoxin reductase , nuclear magnetic resonance , thioredoxin , electrochemistry , electrode , physics
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.