Ping-Pong Protons: How Hydrogen-Bonding Networks Facilitate Heterolytic Bond Cleavage in Peptide Radical Cations

Abstract

Electron capture and electron transfer dissociation (ECD/ETD) tandem mass spectrometry (MS/MS) are commonly employed techniques for biomolecular analysis. The ECD/ETD process predominately cleaves N-Cα peptide backbone bonds, leading to primary sequence information complementary to other mass spectrometry techniques. Despite frequent laboratory use, the mechanistic underpinnings surrounding N-Cα bond cleavage remain debated. While the majority of mechanisms assume a homolytic bond rupture, we recently showed that heterolytic cleavage is also thermodynamically viable. For a cleavage of this type to be feasible, the charge separation created upon breaking of the N-Cα backbone bond must be quickly annihilated. In this work, we show, using density functional computations, that specific hydrogen-bonding motifs and structural rearrangements involving proton transfers stabilize the transition state associated with heterolytic cleavage and eliminate the ensuing charge separation from the final product fragments. The movement of protons can occur either directly from the z- to c-fragment or in a more complex manner including a ping-pong-type mechanism. The nature of these diverse hydrogen-bonding motifs reveals that not only those functional groups proximate to the bond rupture site, but also the entire global chemical environment, play important roles in backbone cleavage characteristic of ECD/ETD MS/MS. For doubly charged systems, both conformation and electron localization site dictate which of the two fragments retains the final positive charge.

N–Cα Bond Cleavage of Zinc-Polyhistidine Complexes in Electron Transfer Dissociation Mediated by Zwitterion Formation: Experimental Evidence and Theoretical Analysis of the Utah–Washington Model

“…In the case of protonated molecules, the intermediates formed by the N−C α bond cleavage can undergo multistep interfragment proton transfers, which stabilize the resulting fragments. 9 Consequently, we considered the formation process of [c′ 2(amide) − H + Zn] + by interfragment proton transfer. As shown in Scheme 2, proton transfer between the iminoenol c′ 2 anion and z • 1 in 2c •+ formed complex 2e •+ , consisting of the amide c′ 2 and the iminoenol z • 1 anion.…”

“…Subsequently, an interfragment proton transfer would occur to produce a protonated c′ fragment and a z • radical. 8,9 Notably, the "Cornell" and "enol" mechanisms are supported by computational studies on c′ and z • fragment formation in model peptides, containing an ammonium radical at the side-chain of lysine residues. 7,10,11 These studies demonstrate that the intermolecular hydrogen transfer from a peptide ammonium radical requires a low-energy barrier to form an aminoketyl radical.…”

Radical solutions: Principles and application of electron‐based dissociation in mass spectrometry‐based analysis of protein structure

“…Mechanisms that assume cleavage on the N‐terminal side of the amide group that the unpaired electron interacts with have also been proposed, initially in 2007 by Zubarev and coworkers and then in 2010 by Tureček et al Some experimental evidence for this N‐terminal cleavage was reported in 2009 by Tsybin and coworkers; however, for small peptides at least, experimental and computational research raised doubts about the feasibility of this mechanism. On the other hand, in several recent studies, Tsybin and coworkers used combined computational and experimental methods to argue that this “enol” mechanism is in many cases thermodynamically and kinetically favored . Because the peptide conformation is important to determine reaction kinetics, it is possible that peptide size and amino acid composition—in particular the type(s) of residue that carry charge—play a role to determine which mechanism dominates, and it is clear that further research is required in this very active field of research.…”

“…On the other hand, in several recent studies, Tsybin and coworkers used combined computational and experimental methods to argue that this "enol" mechanism is in many cases thermodynamically and kinetically favored. [56][57][58] Because the peptide conformation is important to determine reaction kinetics, it is possible that peptide size and amino acid composition-in particular the type(s) of residue that carry charge 40,59 -play a role to determine which mechanism dominates, and it is clear that further research is required in this very active field of research.…”

New Insights on Hydrogen-Bond-Induced Fluorescence Quenching Mechanism of C102–Phenol Complex via Proton Coupled Electron Transfer

“…Intermolecular hydrogen bonding (H-bonding or HB) between a solute (e.g., a fluorophore) and its neighbor (e.g., solvent) may greatly influence the chemical and photochemical behavior of the solute. − For example, H bond-mediated electronic coupling may trigger photoinduced electron transfer between a donor and an acceptor molecule. − An intermolecular H-bonded complex may reorganize in the excited state in such a way that may induce new relaxation pathways which are otherwise unlikely. Thus, the electronic structure of the H-bonded complex and subsequent evolution of its energy in the excited state after photoexcitation is of central importance for molecular level understanding of H-bond-driven photophysical processes.…”