Spectroscopic and Functional Characterization of Iron-Bound Forms of Azotobacter vinelandii^NifIscA

Abstract

The ability of Azotobacter vinelandii NifIscA 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, CD and VTMCD, EPR, 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 ± 1.5cm−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 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.

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

“…Although IscA and its homologs have been characterized previously as alternative scaffold proteins or carriers (15,(37)(38)(39), IscA has a unique and strong iron-binding activity in vitro (40 -44) and in vivo (31) under aerobic conditions. The three invariant cysteine residues (45), and possibly an oxygen ligand (43), are likely involved in iron binding in IscA. Furthermore, the iron center in IscA can be readily mobilized by L-cysteine and transferred for iron-sulfur cluster assembly in vitro under aerobic conditions (43,44,47).…”

“…The three invariant cysteine residues (45), and possibly an oxygen ligand (43), are likely involved in iron binding in IscA. Furthermore, the iron center in IscA can be readily mobilized by L-cysteine and transferred for iron-sulfur cluster assembly in vitro under aerobic conditions (43,44,47). Recently, we also reported that E. coli IscA has its unique activity to bind copper in vivo and in vitro and that excess copper can compete with iron for the metal binding sites in IscA and block iron-sulfur cluster biogenesis (60).…”

“…The iron center in IscA can be readily mobilized by L-cysteine (43,44,47) and transferred for iron-sulfur cluster assembly in proteins in vitro (43,48). These results led us to postulate that IscA may recruit intracellular iron and deliver iron for iron-sulfur cluster biogenesis.…”

Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

“…Three different roles have been assigned to the A. vinelandii IscN counterpart, Nif IscA. First, Nif IscA was shown to bind Fe(II) and Fe(III) and to act as an iron donor for Fe-S biosynthesis (37). Second, Nif IscA was proposed to function as an alternate scaffold for Fe-S biosynthesis (38).…”

Iron–sulfur cluster biogenesis and trafficking in mitochondria

“…1 and 2) (78, 80 -82). The two Isa proteins belong to the A-type family of ISC proteins that have been shown to be either homo-or heterodimers, which can coordinate Fe/S clusters and/or mononuclear iron via three conserved cysteine residues (78,(82)(83)(84). In vitro reconstitution of [2Fe-2S] cluster release from Grx5 to a Isa1-Isa2 heterodimer and subsequent DTT-dependent reductive coupling of two [2Fe-2S] clusters to form a [4Fe-4S] cluster have been reported based on NMR and UV-visible studies (Fig.…”