DIRECT ELECTROCHEMISTRY AND EPR SPECTROSCOPY OF SPINACH FERREDOXIN MUTANTS WITH MODIFIED ELECTRON-TRANSFER PROPERTIES

Mutations of the conserved residue Glu-92 to lysine, glutamine, and al anine have been performed in the recombinant ferredoxin I of spinach l eaves. The purified ferredoxin mutants were found twice as active with respect to,wild-type protein in the NADPH-cytochrome c reductase reac tion catalyzed by ferredoxin-NADP(+) reductase in the presence of ferr edoxin. Cyclic voltammetry and EPR measurements showed that the mutati ons cause a change in the [2Fe-2S] cluster geometry, whose redox poten tial becomes approximately 80 mV less negative. These data point to a role of the Glu-92 side-chain in determining the low redox potential t ypical of the [2Fe-2S] cluster of chloroplast and cyanobacterial ferre doxins, Also a ferredoxin/ferredoxin-NADP(+) reductase chimeric protei n obtained by gene fusion was overproduced in Escherichia coli and pur ified. Fusion of the ferredoxin with its reductase causes only minor e ffects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements.