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

Abstract

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2](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.

Geographic distribution of cadmium and its interaction with the microbial community in the Longjiang River: risk evaluation after a shocking pollution accident

“…There are two important protein types in Desulfovibrio . The first is orange protein (ORP, 11.8 kDa), which contains a mixed metal sulphide cluster of the type [S 2 MoS 2 CuS 2 MoS 2 ] 3− noncovalently bound to the polypeptide chain 59 . The second is cobalt- and zinc-containing adenylate kinases (AKs) 60 .…”

CHAPTER 1. Molybdenum and Tungsten-Containing Enzymes: An Overview

“…Besides the molybdenum and tungsten centres that form the active site of several enzymes (described in the previous sections), there are also a few other heteronuclear centres whose physiological function is not yet fully understood. [669][670][671][672][673] X-ray absorption fine structure (eXaFs) studies have revealed the presence of a novel linear cluster formulated as [s 2 Mos 2 Cus 2 Mos 2 ] ( Figure 1.3b), a unique heterometallic cluster in biological systems. 669,670 the cluster is contained in a small monomeric "orange protein" isolated from sulfate reducing bacteria, and is non-covalently bound to the protein.…”

Progress toward the Isolation of Late Metal Terminal Sulfides

“…[18] Finally, the orange protein (ORP) features a Cu + ion coordinated to four sulfide ligands in a tetrahedral array. [19,20] While the function of ORP is still unknown, it has been proposed to function as a Cu-storage protein. [21] Sulfide also plays an important structural role in the Nicontaining enzymes, NiFe dehydrogenase and acetyl-CoA synthase.…”