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

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, 356and 279nm, an intense EPR resonance with a g value around 4.3 and Mössbauer spectroscopy parameters of δ equal to 0.69mm/s and ΔEQ equal to 3.25mm/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 191nm and minima at 203 and 231nm. Most significantly, the presence of isosbestic points at 189 and 228nm 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.