TitleSpectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.
Publication TypeJournal Article
Year of Publication2012
AuthorsMapolelo, DT, Zhang, B, Naik, SG, Huynh, BHanh, Johnson, MK
Date Published2012 Oct 16
KeywordsAzotobacter vinelandii, Bacterial Proteins, Circular Dichroism, Electron Spin Resonance Spectroscopy, iron, Oxidation-Reduction, Polymerase Chain Reaction, Spectrophotometry, Ultraviolet

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.

Alternate JournalBiochemistry
PubMed ID23003563
PubMed Central IDPMC3546131
Grant ListGM47295 / GM / NIGMS NIH HHS / United States
GM62524 / GM / NIGMS NIH HHS / United States
R01 GM047295 / GM / NIGMS NIH HHS / United States
R01 GM062524 / GM / NIGMS NIH HHS / United States
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