title: Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties.
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.
authors.affiliation: Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Aliverti
authors.forename: W R
authors.initials: WR
authors.lastname: Hagen
authors.forename: G
authors.initials: G
authors.lastname: Zanetti
mesh_terms: D004553:Electric Conductivity; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000502:physiology; D009154:Mutation / Q000502:physiology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018724:Spinacia oleracea / Q000737:chemistry
chemical_list: D005288:Ferredoxins; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D009251:NADPH-Ferrihemoprotein Reductase