Locating Pb2+ and Zn2+ in zinc finger-like peptides using mass spectrometry.

The binding preferences of Pb(2+)and Zn(2+) in doubly charged complexes with zinc finger-like 12-residue peptides (Pep), [Mn(Pep-2(n-1)H)](2+) have been explored using tandem mass spectrometry. The peptides were synthesized strategically by blocking the N-terminus with an acetyl group and with four cysteine and/or histidine residues in positions 2, 5, 8, and 11, arranged in different motifs: CCHH, CHCH, and CCCC. The MS(2) spectra of the Pb(2+) and Zn(2+) complexes show multiple losses of water and a single methane loss and these provide a sensitive method for locating the metal dication and so elucidating its coordination. The elimination of a methane molecule indicated the position of the metal at the Cys2 residue. Whereas lead was observed to preferentially bind to cysteine residues, zinc was found to primarily bind to histidine residues and secondarily to cysteine residues. Preferential binding of lead to cysteine is preserved in the complexes with more than one Pb(2+). Key to the mechanism of the loss of water and methane is the metal dication withdrawing electrons from the proximal amidic nitrogen. This acidic nitrogen loses its hydrogen to an amidic oxygen situated four atoms away leading to formation of a five-member ring and the elimination of water.

Lead(II)-catalyzed oxidation of guanine in solution studied with electrospray ionization mass spectrometry.

The oxidation of guanine was investigated in water/methanol solution both in the absence and in the presence of Pb(II) with a variable temperature reactor coupled to a tandem mass spectrometer that allowed signature ions of solution reagents and products to be monitored by electrospray ionization (ESI). Two different oxidizing agents were employed, one strong (peroxymonosulfuric acid) and one weaker (hydrogen peroxide). Peroxymonosulfuric acid was observed to oxidize guanine rapidly at room temperature, k(app) > 10(-2) s(-1), whether in the absence or in the presence of Pb(II), to produce spiroiminohydantoin. Guanine did not show measurable oxidation by hydrogen peroxide in the absence of Pb(II) at concentrations of H(2)O(2) up to 1 M at temperatures up to 333 K (k(app) < 3 × 10(-8) s(-1) at 298 K), but in the presence of Pb(II), it was observed to produce both 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) and imidazolone (Iz) in a ratio of 2.3 ± 0.1 with a total rate enhancement of more than 4 × 10(3). The activation energy was measured to be 82 ± 11 kJ mol(-1) and is more than 120 kJ mol(-1) lower than that for the uncatalyzed oxidation with hydrogen peroxide measured to be at least 208 ± 26 kJ mol(-1). An activation energy of 113 ± 9 kJ mol(-1) has been reported by Bruskov et al. (Nucleic Acids Res.2002, 30, 1354) for the heat-induced oxidation by hydrogen peroxide of guanine embedded as guanosine in DNA which leads to the production of 8-oxo-7,8-dihydro-guanine (8-oxo-Gua). The atomic lead dication lowers the activation energy by activating the hydrogen peroxide oxidant, possibly by O-O bond activation, and by directing the oxidation, possibly through coordination to the functional groups adjacent to the carbon C5: the C6 carbonyl group and the N7 nitrogen. The coupling of tandem mass spectrometry (MS(2)) with a simple variable temperature reactor by ESI proved to be very effective for measuring reaction kinetics and activation energies in solution. Signature ions of both reagents and products, as well as the catalyst, could be identified, and the data were acquired in real time. The technique should be suitable for exploring other chemical and biochemical reactions that occur on similar time scales (minutes to hours).

Structure of [Pb(Gly-H)]+ and the monosolvated water and methanol solvated species by infrared multiple-photon dissociation spectroscopy, energy-resolved collision-induced dissociation, and electronic structure calculations.

Infrared multiple-photon dissociation (IRMPD) spectroscopy, collision-induced dissociation mass spectrometry, and theoretical calculations are combined to provide new insights into the structure and dissociation of lead(II) complexed with the conjugate acid of the amino acid glycine ([Pb(Gly-H)](+)) in the presence and absence of solvent. Unexpectedly, these experiments show the main site of lead(II) coordination to be the deprotonated amino group of glycine, with additional coordination to the carbonyl group. In such a structure lead(II) can act as an effective conduit for proton/hydrogen shifts, making H(2)O loss competitive with that of CO in the [Pb(Gly-H)](+) complex and leading to solvent deprotonation and formation of [PbOR(Gly)](+) (R = H, CH(3)) ions when solvent is present in the complex. The structural assignments based on IRMPD spectroscopy are complemented with isotopic labeling experiments (H(2)(18)O) and experiments done on the ethyl ester of glycine.