ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P27. Orange Protein Mo-Cu Cluster Reconstitution
M.S. Carepo a , S.R. Pauleta a , A. Duarte a , D.S. Figueiredo a , J.G. Graff a , A.G. Wedd b ,
A.S. Pereira a , J.J.G. Moura a , I. Moura a
a
Requimte – Departamento de Quimica, CQFB, Faculdade de Ciências e Tecnologia – UNL, 2825 Monte da
Caparica, Portugal
e-mail: marta.carepo@dq.fct.unl.pt
b
School of Chemistry, University of Melbourne, ParkVille, Victoria 3010, Australia
The orange protein (ORP) isolated from Desulfovibrio gigas is a monomeric protein of approximately 12 kDa.
X-ray absorption fine structure (EXAFS) studies revealed the presence of a mixed-metal sulfide linear cluster
[S2MoS2CuS2MoS2] 3- which is a quite unusual protein bound heterometallic cluster [1,2]. Tetrathiomolybdate
and copper are known to form sulphide metal complexes and their antagonism has been exploited for the
treatment of Wilson’s disease and breast cancer by dietary supplement on tetrathiomolybdate [3].
ORP was heterologously expressed in E. coli as an apo-protein, the holo protein can be reconstitute in vitro by
the addition of copper and tetrathiomolybdate (TM4) or tetrathiotungstate (TW4). The apo-ORP reconstitution
using TM4 and Cu 2+ (4Mo:2Cu/ protein) is dependent on the incubation order of the metals with ORP. When
TM4 is added to ORP followed by the addition of Cu 2+ the metal ratio obtained is 1Mo:1Cu the protein whereas
when Cu 2+ is first incubated with ORP followed by TM4 addition the metal ratio is 2Mo:1Cu, as observed for the
native protein. The percentage of reconstitution was determined by 1H-15N HSQC.
In this work we present UV-visible titrations of (ORP+TM4) with Cu 2+ and (ORP+ Cu 2+ ) with TM4. Different
stages of the interaction between ORP and the metals to form de metal cluster were followed by EPR.
The two metals were also allowed to react first and then the reaction mixture was incubated with the protein.
Titrations of the two metals in the absence of ORP were also performed in order to investigate the role of ORP in
the cluster formation. The results obtained can give some insights concerning the ORP cluster assembling in the
native protein.
Acknowledgement: We thank Fundação para a Ciência e a Tecnologia for financial support
References:
[1] G.N. George, I. J. Pickering, E. Y. Yu, R.C. Prince, S.A. Bursakov, O.Y. Gavel, J.J.G. Moura and
I. Moura, JACS, 122, 8321, (2000).
[2] S.A. Bursakov, O.Y. Gavel, G. Di Rocco, J. Lampreia, J. Calvete, A.S. Pereira, J.J.G. Moura and
I. Moura, J Inorg Biochem, 98, 833 (2004).
[3] H. Laurie, Eur. J. Inorg. Chem., 2000, 2443 (2000).
_____________________________________________________________________
146