TY - JOUR
T1 - Structural features and stability of apo- and holo-forms of a simple iron–sulfur protein
AU - Almeida, Ana V.
AU - Jacinto, João P.
AU - Guerra, João P.L.
AU - Vieira, Bruno J.C.
AU - Waerenborgh, João C.
AU - Jones, Nykola C.
AU - Hoffmann, Søren V.
AU - Pereira, Alice S.
AU - Tavares, Pedro
N1 - Funding Information:
This research was supported by Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES), grant PTDC/BIA-PRO/111485/2009 (to P.T.), PTDC/QUI/64248/2006 (to A.S.P). This work was also supported by Radiation Biology and Biophysics Doctoral Training Programme (RaBBiT, PD/00193/2012, Applied Molecular Biosciences Unit – UCIBIO (UIDB/04378/2020), CEFITEC (UIDB/00068/2020) and C2TN (UID/Multi/04349/2019), all financed by national funds from FCT-MCTES). A.V.A. (PD/BD/135477/2017), J.P.J. (SFRH/BD/135056/2017), and J.P.L.G. (PD/BD/135476/2017) are supported by the RaBBiT programme. This work benefited from STSM funding by COST Action (CA15126 MOBIEU) and by the project CALIPSOplus under the Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020.
Publisher Copyright:
© 2021, European Biophysical Societies' Association.
PY - 2021/5
Y1 - 2021/5
N2 - 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.
AB - 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.
KW - Iron–sulfur proteins
KW - Protein secondary structure
KW - Rubredoxin
KW - Synchrotron radiation circular dichroism
UR - http://www.scopus.com/inward/record.url?scp=85105979529&partnerID=8YFLogxK
U2 - 10.1007/s00249-021-01546-0
DO - 10.1007/s00249-021-01546-0
M3 - Article
C2 - 34009405
AN - SCOPUS:85105979529
VL - 50
SP - 561
EP - 570
JO - European Biophysics Journal With Biophysics Letters
JF - European Biophysics Journal With Biophysics Letters
SN - 0175-7571
IS - 3-4
ER -