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
JournalBiochemistry
Volume51
Issue41
Pagination8056-70
Date Published2012 Oct 16
ISSN1520-4995
KeywordsAzotobacter vinelandii, Bacterial Proteins, Circular Dichroism, Electron Spin Resonance Spectroscopy, iron, Oxidation-Reduction, Polymerase Chain Reaction, Spectrophotometry, Ultraviolet
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.

DOI10.1021/bi300664j
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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