title: Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.
abstract: The ability of Azotobacter vinelandii(Nif)IscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe-S cluster biogenesis. (Nif)IscA is shown to bind one Fe(III) or one Fe(II) per homodimer and the spectroscopic and redox properties of both the Fe(III)- and Fe(II)-bound forms have been characterized using the UV-visible absorption, circular dichroism, and variable-temperature magnetic circular dichroism, electron paramagnetic resonance, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (S = 3/2) Fe(III) center (E/D = 0.33, D = 3.5 ± 1.5 cm(-1)) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (S = 2) Fe(II) center (E/D = 0.28, D = 7.6 cm(-1)) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound (Nif)IscA undergoes reversible redox cycling between the Fe(III)/Fe(II) forms with a midpoint potential of +36 ± 15 mV at pH 7.8 (versus NHE). l-Cysteine is effective in mediating release of free Fe(II) from both the Fe(II)- and Fe(III)-bound forms of (Nif)IscA. Fe(III)-bound (Nif)IscA was also shown to be a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results.
authors.affiliation: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA.
authors.forename: Daphne T
authors.initials: DT
authors.lastname: Mapolelo
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Sunil G
authors.initials: SG
authors.lastname: Naik
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D007501:Iron