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

Abstract

Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [SMoSCuSMoS] (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of Cu plus tetrathiomolybdate to apo-ORP under controlled conditions. In the work described herein, we artificially inserted the ATCUN ("amino terminus Cu and Ni") motif in the Desulfovibrio gigas ORP (AlaSerHis followed by the native amino acid residues; modified protein abbreviated as ORP*) to increase our understanding of the Mo/Cu cluster assembly in ORP. The apo-ORP* binds Cu in a 1:1 ratio to yield Cu-ORP*, as clearly demonstrated by EPR (g = 2.183, 2.042 and A = 207 × 10 cm, 19 × 10 cm) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ε = 90 M cm). The H NMR spectrum shows that His and His are significantly affected upon the addition of the Cu. The X-ray structure shows that these two residues are very far apart (C-C ≈ 27.9 Å), leading us to suggest that the metal-induced NMR perturbations are due to the interaction of two protein molecules with a single metal ion. Docking analysis supports the metal-mediated dimer formation. The subsequent tetrathiomolybdate binding, to yield the native Mo/Cu cluster, occurs only upon addition of dithiothreitol, as shown by UV-visible and NMR spectroscopies. Additionally, H NMR of Ag-ORP* (Ag used as a surrogate of Cu) showed that Ag strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that Cu and Cu have two different binding sites in ORP*. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that Cu is reduced to Cu and transferred from the ATCUN motif to the methionine site; finally, Cu is transferred to the cluster-binding region, upon the interaction of two protein molecules. This result may suggest that copper trafficking is triggered by redox-dependent coordination properties of copper in a trafficking pathway.

NiII‐ATCUN‐Catalyzed Tyrosine Nitration in the Presence of Nitrite and Sulfite

“…[27] Peak vi sa lso attributedt ot he -S(O)-CH 3 methyl group, but could only be seen in oxidized apo-ATCUN-ORP ( Figure S2 in the SupportingI nformation;n ot in oxidized Ni-ATCUN-ORP). [20] Besides the use of H 2 O 2 ,i nt his study,N i II -ATCUN-ORP was oxidizedinthe presence of SO 3 2À /O 2 and the UV/Vis absorption spectrum shows ac harge-transferb and at approximately 372 nm ( Figure S4, Supporting Information) that is similar to the H 2 O 2 oxidation method and also other reported N iII -peptide with sulfite oxidation. [28] The sulfite autooxidation by Ni II -ATCUN-ORP produce reactive oxysulfur radicals.…”

“…To the best of our knowledge, this is the first report of 3‐NT formation by a Ni II ‐ATCUN in the presence of the NO 2 − /SO 3 2− /O 2 system. For this experiment, we used fusion orange protein (ATCUN‐ORP) as an in vitro model complex containing ATCUN tag at the N‐terminus (ASH) and native Tyr 75 located in cluster binding region of ATCUN‐ORP (Figure ) . Recently, we have reported that cluster formation in ORP occur by protein–protein interaction in head‐to‐tail fashion, in which the N‐terminus metal‐binding ATCUN motif comes to the cluster‐binding region, in which Tyr residues are affected through pseudocontact .…”

“…The v a and v b signals are assigned to the methyl protons of ‐S(O)‐CH 3 of the R and S forms . Peak v is also attributed to the ‐S(O)‐CH 3 methyl group, but could only be seen in oxidized apo‐ATCUN‐ORP (Figure S2 in the Supporting Information; not in oxidized Ni‐ATCUN‐ORP) …”

“…[22] Peak iii at 6.85 ppm (doublet) may correspondt othe H d /H e of Tyr 75 ,according to previousN MR data; [23] this means that the Ni II -ATCUN motif in Ni II -ATCUN-ORP interacts with the aromatic group of the Tyrr esidue. In the aliphatic region, only the alanine site resonances at 1.310 and 1.298 ppm are highly affected (not shown) in as imilarm anner to Cu-ATCUN-ORP, [20] but the Met signals at 1.987 and 1.890 ppm are not affected.…”

“…[20,21] Recently,w eh ave reported that cluster formation in ORP occur by protein-protein interaction in head-to-tail fashion, in which the N-terminus metal-binding ATCUN motif comes to the cluster-binding region, in whichT yr residues are affected through pseudocontact. [20] Therefore, taking advantage of this model system, Ni II -ATCUN-ORP was synthesized in the presence of NiCl 2 and apo-ATCUN-ORP at 1:1r atio and characterized by UV/Vis absorption and 1 HNMR spectroscopic studies. The Ni II -ATCUN-ORP can be oxidizedb yt he H 2 O 2 or SO 3 2À /O 2 system and, subsequently, produce HOC or oxysulfur radicals, respectively,w hich are evaluated by EPR spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap.…”