Structural features and stability of apo- and holo-forms of a simple iron–sulfur protein

Ana V. Almeida, João P. Jacinto, João P.L. Guerra, Bruno J.C. Vieira, João C. Waerenborgh, Nykola C. Jones, Søren V. Hoffmann, Alice S. Pereira, Pedro Tavares

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Iron–sulfur centers are widespread in living organisms, mostly performing electron transfer functions, either in electron transfer chains or as part of multi-enzymatic complexes, while being also present in enzyme active sites, handling substrate catalysis. Rubredoxin is the simplest iron–sulfur containing protein constituted by a single polypeptide chain of 50 to 60 amino acids, of which four cysteine residues are responsible for metal binding in a tetrahedral coordination sphere. In this manuscript we explore the structure and stability of both apo- and holo-forms of a Rubredoxin from Marinobacter hydrocarbonoclasticus using Synchrotron Radiation Circular Dichroism (SRCD) in combination with other biochemical and spectroscopic techniques. The results are consistent with a holo-protein form containing a monomeric iron center with UV–visible maxima at 760, 578, 494, 386, 356 and 279 nm, an intense EPR resonance with a g value around 4.3 and Mössbauer spectroscopy parameters of δ equal to 0.69 mm/s and ΔEQ equal to 3.25 mm/s, for the ferrous reconstituted state. SRCD data, obtained for the first time for the apo-form, show a quite defined structure with ∆ε maximum at 191 nm and minima at 203 and 231 nm. Most significantly, the presence of isosbestic points at 189 and 228 nm made the interconversion between the two stable apo- and holo-form solution structures clear. SRCD temperature dependence data shows that for both forms the denaturation process proceeds through an intermediate species.

Original languageEnglish
Pages (from-to)561-570
Number of pages10
JournalEuropean Biophysics Journal
Volume50
Issue number3-4
DOIs
Publication statusPublished - May 2021

Keywords

  • Iron–sulfur proteins
  • Protein secondary structure
  • Rubredoxin
  • Synchrotron radiation circular dichroism

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