ESI-IM-MS reveals the metal binding of three analog methanobactin peptides with different numbers of free Cys at physiological pH

Abstract

Three analog methanobactin (amb7) peptides containing the primary structure acetyl-Leu1-His2-Cys3-Gly4-Ser5-Cys6-Tyr7-Pro8-His9-Cys10-Ser11-Cys12-Met13 but differing by the number of disulfide bridges, have been studied for their reactivity with Fe(III), Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Ag(I), and Pb(II) using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) and fluorescence quenching. The Pb(II) bound only with the amb7-0 (0 disulfide bonds), Zn(II) only to amb7-0 and amb7-1 (1 disulfide bond), Ni(II) only to amb7-1 and amb7-2 (2 disulfide bonds), and Cu(II) to amb7-2, concomitant with the oxidation of amb7-0 and amb7-1. The reduction of Cu(II) to Cu(I) resulted with 1-4Cu(I) binding to amb7-1 and amb7-0, while Ag(I) also exhibited 1-6 Ag(I) ions binding through a mechanism indicative of labile proton exchange. The binding of the oxidized Co(III/V) and Mn(III/V) were observed at pH 9 and 11, while Fe(III) did not exhibit significant binding. The observed negatively charged complexes were consistent with the deprotonation of the metal sites of Cys, His, carboxyl terminus and indicative of ion-ion interactions with the metal at pH 7, 9, and 11. The positively charged complexes were indicative of ion-dipole metal binding but at pH 9 and pH 11 gave results similar to those of the negative ion analyses. Fluorescence quenching also qualitatively agreed with the ESI-IM-MS results providing evidence that they revealed the solution-phase metal binding behavior of amb7.