Mo–Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S 2 MoS 2 CuS 2 MoS 2 ] 3- noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the ap...

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Bibliographic Details
Published in:Journal of biological inorganic chemistry 2014-06, Vol.19 (4-5), p.605-614
Main Authors: Carepo, Marta S. P., Pauleta, Sofia R., Wedd, Anthony G., Moura, José J. G., Moura, Isabel
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Biochemistry
Biomedical and Life Sciences
Copper - chemistry
Copper - metabolism
Desulfovibrio gigas
Desulfovibrio gigas - chemistry
Desulfovibrio gigas - metabolism
Escherichia coli
Life Sciences
Microbiology
Molybdenum - chemistry
Molybdenum - metabolism
Original Paper
Topical Issue in honor of Ivano Bertini
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Summary:The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S 2 MoS 2 CuS 2 MoS 2 ] 3- noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The 1 H– 15 N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.
ISSN:0949-8257
1432-1327
DOI:10.1007/s00775-014-1107-8