The pH Dependent Cu(II) and Zn(II) Binding Behavior of an Analog Methanobactin Peptide

Abstract

The pH dependent reactivity of an analog methanobactin peptide (amb) with the sequence acetyl-His1-Cys2-Gly3-Pro4-His5-Cys6 (Mw = 694.79 Da) was investigated for its binding ability for a series of biologically active metal ions using ion mobility-mass spectrometry. Cu(II), Zn(II) and, to a lesser extent, Ni(II) were observed to form complexes with amb from 1 : 1 molar equivalent amb:metal(II) solutions at pH > 6, indicating the deprotonation of the imidazole N of His (pKa = 6.0) must occur to allow the initial anchoring of the metal(II) ion. The amb-metal(II) complexes were observed as both positive and negative ions, although the Zn(II) complexes preferred forming an overall negative ion complex which is consistent with the two thiolate groups of Cys2 and Cys6 being involved in Zn(II) coordination. The Cu(II) addition, however, always resulted in a Cys-Cys disulfide bridge in both Cu-free amb and Cu-bound amb, which excluded thiolate coordination to Cu(II). Collision cross- section measurements showed the Zn(II) and Cu(II) negative ion complexes were smaller than the positive ion complexes, suggesting Zn(II) binds most compactly via the imidazole N of His and the thiolate groups of Cys, whereas Cu(II) binds most compactly via the imidazole N of His and two deprotonated N of two amide groups on the peptide backbone. The lowest energy structures from the B3LYP/LanL2DZ level of theory showed the functional groups of His5, Cys2 and Cys6 coordinated to Zn(II), whereas the His1 and the amide nitrogens of Cys2 and Gly3 coordinated to Cu(II), producing an overall negative charged complex. The positive ion complexes of Zn(II) and Cu(II) were both shown to coordinate via the two imidazole nitrogens of His1 and His5 and either the oxygen of the backbone carbonyl of Cys6 or the oxygen of the C-terminal, respectively.

Redox activity and multiple copper(I) coordination of 2His–2Cys oligopeptide

“…A previous study on the pH‐dependent Cu(II) and Zn(II) binding behavior of analog methanobactin peptide‐1 (amb 1 ), with the sequence acetyl–His 1 –Cys 2 –Gly 3 –Pro 4 –His 5 –Cys 6 (MW = 694.81 Da) showed both Cu(II) and Zn(II) bound to amb 1 above pH 6.0, but by different mechanisms. Cu(II) addition led to the oxidation of amb 1 and Cu(I) binding below pH 6.0.…”

Comparison of the pH‐dependent formation of His and Cys heptapeptide complexes of nickel(II), copper(II), and zinc(II) as determined by ion mobility‐mass spectrometry

“…The amb 5 Mass Spectrometer (G1) 51 with ESI conditions and traveling wave settings optimized to limit structural rearrangement and the dissociation of the amb species. Typical operating conditions are described in the Supporting Information.…”

“…The formation of complexes between Ni(II) and A to F was most predominantly observed as negatively-charged species. Figure 2 shows that the free [amb 5 -H] − was the main species at pH ≤ 7.0, for all amb 5A-F species, and that A, B, and C formed the highest percentage of negative ion Ni(II) complexes over pH ≥ 8. Our previous study 2 of A to F located low energy B3LYP/LanL2DZ conformers of [amb 5 -H] − that were stabilized by the Cys thiolate and C-terminus carboxylate salt-bridged or H-bonded to the imidazolium or imidazole groups of His.…”

“…Our previous IM‐MS studies of metal binding have shown behavior that is closely correlated to that of the solution phase, because the covalent interactions between the metal ion and peptide are robust enough to be conserved during electrospray ionization (ESI). Previous studies included the methanobactin from Methylosinus trichosporium (mb‐OB3b) and analog methanobactin (amb) oligopeptides which replaced the two enethiol oxazolone binding sites of mb‐OB3b with the 2His‐2Cys binding motif . From solution‐phase chemistry, the amb 5A‐F (Figure A ‐ F ) would be expected to exhibit an overall charge based on the protonation state of their acidic and basic sites, ie, the C‐terminus carboxyl group (p K a ≈ 3), His (p K a ≈ 6.0), Cys (p K a ≈ 8.3), and Tyr (p K a ≈11.0) .…”

“…Previous studies included the methanobactin 3,4 from Methylosinus trichosporium (mb-OB3b) and analog methanobactin (amb) oligopeptides which replaced the two enethiol oxazolone binding sites of mb-OB3b with the 2His-2Cys binding motif. 1,[5][6][7] From solution-phase chemistry, the amb 5A-F ( Figure 1A-1F) would be expected to exhibit an overall charge based on the protonation state of their acidic and basic sites, ie, the Cterminus carboxyl group (pKa ≈ 3), His (pKa ≈ 6.0), Cys (pKa ≈ 8.3),…”