Orange protein from Desulfovibrio alaskensis G20: insights into the Mo–Cu cluster protein-assisted synthesis

Abstract

A novel metalloprotein containing a unique [S2MoS2CuS2MoS2](3-) cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI-MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S-Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV-visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.

Insights into the Molybdenum/Copper Heterometallic Cluster Assembly in the Orange Protein: Probing Intermolecular Interactions with an Artificial Metal-Binding ATCUN Tag

“…The metal cluster, [Ag I (MoS 4 ) 2 ] 3− , is reconstituted in the recombinant protein employing a procedure similar to that described for Mo/Cu. 4 First, 1 mM AgNO 3 was added into 1 mM apo-ORP*, in 50 mM HEPES, pH 7.5, followed by the addition of 2 mM (NH 4 ) 2 MoS 4 ; the mixture was incubated for 30 min in an ice bath (4 °C). The protein was subsequently separated from unbounded, small molecules (metal ions and TTM) by size-exclusion chromatography (PD-10 column; size exclusion of 5 kDa; GE Healthcare) equilibrated with 50 mM 35 where key methionine and histidine residues are highlighted (with ORP* numbering), as well as the distances measured between sulfur atoms (golden sphere) and C α atoms (gray sphere).…”

“…The metal cluster, [Ag I (MoS 4 ) 2 ] 3– , is reconstituted in the recombinant protein employing a procedure similar to that described for Mo/Cu . First, 1 mM AgNO 3 was added into 1 mM apo-ORP*, in 50 mM HEPES, pH 7.5, followed by the addition of 2 mM (NH 4 ) 2 MoS 4 ; the mixture was incubated for 30 min in an ice bath (4 °C).…”

“…Recently, we also described a simple, straightforward method for reconstitution of other recombinant ORPs from Desulfovibrio alaskensis G20, where the ATCUN tag was absent. 4 In this simple process, Cu II (free from ATCUN) is first reduced by TTM (cysteine is absent in both D. alaskensis and D. gigas), and finally TTM binds Cu I to yield the native Mo/Cu cluster.…”

“…Orange protein (ORP), first isolated from the sulfate-reducing organism Desulfovibrio gigas , is found in several anaerobic bacteria. − It harbors a unique molybdenum/copper heterometallic cluster, [S 2 Mo VI ­S 2 Cu I ­S 2 Mo VI S 2 ] 3– (Mo/Cu), revealed by EXAFS (Figure A) . The metal cofactor is stabilized in the binding pocket through non-covalent interactions, but it does not involve any of the common metal-binding amino acid side chains, such as cysteine, histidine, and methionine side chains.…”

“…The crystal structure of apo-ORP* was previously determined (Figure B). ,, The reconstitution of the Mo/Cu cluster in ORP* from D. gigas was not straightforward, and later it was modified by addition of DTT, but the role of the metal-binding properties of this small ATCUN tag was overlooked. Recently, we also described a simple, straightforward method for reconstitution of other recombinant ORPs from Desulfovibrio alaskensis G20, where the ATCUN tag was absent . In this simple process, Cu II (free from ATCUN) is first reduced by TTM (cysteine is absent in both D. alaskensis and D. gigas ), and finally TTM binds Cu I to yield the native Mo/Cu cluster.…”