title: Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I. Changes in [4Fe-4S] cluster reduction potential and reactivity.
abstract: We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
authors.forename: S E
authors.initials: SE
authors.lastname: Iismaa
authors.forename: A E
authors.initials: AE
authors.lastname: Vázquez
authors.forename: G M
authors.initials: GM
authors.lastname: Jensen
authors.forename: P J
authors.initials: PJ
authors.lastname: Stephens
authors.forename: J N
authors.initials: JN
authors.lastname: Butt
authors.forename: F A
authors.initials: FA
authors.lastname: Armstrong
authors.forename: B K
authors.initials: BK
authors.lastname: Burgess
mesh_terms: D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry
chemical_list: D005288:Ferredoxins; D046911:Macromolecular Substances