Spectroscopic and functional characterization of iron-bound forms of azotobacter vinelandii ^NifIscA

• Type of Material: Article
• Authors: Daphne T. Mapolelo, University of Georgia; Bo Zhang, University of Georgia; Sunil Naik, Emory University; Boi Hanh Huynh, Emory University; Michael K. Johnson, University of Georgia
• Language: English
• Date: 2012-10-16
• Publisher: MAIK Nauka/Interperiodica
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/bi300664j
• Title of Journal or Parent Work: Биохимия / Biochemistry
• ISSN: 0006-2979
• Volume: 51
• Issue: 41
• Start Page: 8056
• End Page: 8070
• Grant/Funding Information: This work was support by grants for the NIH (GM62542 to M.K.J. and GM47295 to B.H.H.)
• Research Categories: Physics, General; Chemistry, Biochemistry

Abstract

The ability of Azotobacter vinelandiiNifIscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe-S cluster biogenesis. NifIscA 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 NifIscA 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 NifIscA. Fe(III)-bound NifIscA 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.