In solution, half-cell potentials are measured relative to those of other half cells, thereby establishing a ladder of thermochemical values that are referenced to the standard hydrogen electrode (SHE), which is arbitrarily assigned a value of exactly 0 V. Although there has been considerable interest in, and efforts toward, establishing an absolute electrochemical half-cell potential in solution, there is no general consensus regarding the best approach to obtain this value. Here, ion-electron recombination energies resulting from electron capture by gas-phase nanodrops containing individual [M(NH3)6]3+, M = Ru, Co, Os, Cr, and Ir, and Cu2+ ions are obtained from the number of water molecules that are lost from the reduced precursors. These experimental data combined with nanodrop solvation energies estimated from Born theory and solution-phase entropies estimated from limited experimental data provide absolute reduction energies for these redox couples in bulk aqueous solution. A key advantage of this approach is that solvent effects well past two solvent shells, that are difficult to model accurately, are included in these experimental measurements. By evaluating these data relative to known solution-phase reduction potentials, an absolute value for the SHE of 4.2 +/- 0.4 V versus a free electron is obtained. Although not achieved here, the uncertainty of this method could potentially be reduced to below 0.1 V, making this an attractive method for establishing an absolute electrochemical scale that bridges solution and gas-phase redox chemistry.

Electrospray ionization (ESI) mass spectrometry (MS) is a crucial method for rapidly determining the interactions between small molecules and proteins with ultrahigh sensitivity. However, nonvolatile molecules and salts that are often necessary to stabilize the native structures of protein–ligand complexes can readily adduct to protein ions, broaden spectral peaks, and lower signal-to-noise ratios in native MS. ESI emitters with narrow tip diameters (∼250 nm) were used to significantly reduce the extent of adduction of salt and nonvolatile molecules to protein complexes to more accurately measure ligand–protein binding constants than by use of conventional larger-bore emitters under these conditions. As a result of decreased salt adduction, peaks corresponding to protein–ligand complexes that differ in relative molecular weight by as low as 0.06% can be readily resolved. For low-molecular-weight anion ligands formed from sodium salts, anion-bound and unbound protein ions that differ in relative mass by 0.2% were completely baseline resolved using nanoscale emitters, which was not possible under these conditions using conventional emitters. Owing to the improved spectral resolution obtained using narrow-bore emitters and an analytically derived equation, K d values were simultaneously obtained for at least six ligands to a single druggable protein target from one spectrum for the first time. This research suggests that ligand–protein binding constants can be directly and accurately measured from solutions with high concentrations of nonvolatile buffers and salts by native MS.

Hydrated divalent magnesium and calcium clusters are used as nanocalorimeters to measure the internal energy deposited into size-selected clusters upon capture of a thermally generated electron. The infrared radiation emitted from the cell and vacuum chamber surfaces as well as from the heated cathode results in some activation of these clusters, but this activation is minimal. No measurable excitation due to inelastic collisions occurs with the low-energy electrons used under these conditions. Two different dissociation pathways are observed for the divalent clusters that capture an electron: loss of water molecules (Pathway I) and loss of an H atom and water molecules (Pathway II). For Ca(H 2 O) n 2ϩ , Pathway I occurs exclusively for n Ն 30 whereas Pathway II occurs exclusively for n Յ 22 with a sharp transition in the branching ratio for these two processes that occurs for n Ϸ 24. The number of water molecules lost by both pathways increases with increasing cluster size reaching a broad maximum between n ϭ 23 and 32, and then decreases for larger clusters. From the number of water molecules that are lost from the reduced cluster, the average and maximum possible internal energy is determined to be ϳ4.4 and 5.2 eV, respectively, for Ca(. This value is approximately the same as the calculated ionization energies of M(H 2 O) n ϩ , M ϭ Mg and Ca, for large n indicating that the vast majority of the recombination energy is partitioned into internal modes of the ion and that the dissociation of these ions is statistical. For smaller clusters, estimates of the dissociation energies for the loss of H and of water molecules are obtained from theory. For Mg(H 2 O) n 2ϩ , n ϭ 4 -6, the average internal energy deposition is estimated to be 4.2-4.6 eV. The maximum possible energy deposited into the n ϭ 5 cluster is Ͻ7.1 eV, which is significantly less than the calculated recombination energy for this cluster. There does not appear to be a significant trend in the internal energy deposition with cluster size whereas the recombination energy is calculated to increase significantly for clusters with fewer than 10 water molecules. These, and other results, indicate that the dissociation of these smaller clusters is nonergodic. . The effectiveness of the bottom-up method for complex samples can be enhanced by using multidimensional separations. Clemmer and coworkers elegantly demonstrated that combining on-line liquid chromatography (LC) with ion mobility spectrometry and MS can greatly improve separations without increasing analysis times over LC/MS alone [2][3][4]. In contrast, the "top-down" approach to protein characterization has the advantage that de novo sequencing, including the identification and structural localization of labile posttranslational modifications, can be done directly on protein mixtures without proteolysis [5,6]. This top-down approach has greatly benefited from the development of electron capture dissociation (ECD), a method pioneered by McLafferty and coworkers [6][7][8][9]. In a typical ECD experim...

The extent of internal energy deposition into ions upon storage, radial ejection, and detection using a linear quadrupole ion trap mass spectrometer is investigated as a function of ion size (m/z 59 to 810) using seven ion-molecule thermometer reactions that have well characterized reaction entropies and enthalpies. The average effective temperatures of the reactants and products of the ion-molecule reactions, which were obtained from ion-molecule equilibrium measurements, range from 295 to 350 K and do not depend significantly on the number of trapped ions, m/z value, ion trap q z value, reaction enthalpy/entropy, or the number of vibrational degrees of freedom for the seven reactions investigated. The average of the effective temperature values obtained for all seven thermometer reactions is 318 ± 23 K, which indicates that linear quadrupole ion trap mass spectrometers can be used to study the structure(s) and reactivity of ions at near ambient temperature.

In solution, half-cell potentials are measured relative to other half-cells resulting in a ladder of thermodynamic values that is anchored to the standard hydrogen electrode (SHE), which is assigned an arbitrary value of exactly 0 V. A new method for measuring the absolute SHE potential is introduced in which reduction energies of Eu(H 2 O) n 3+ , from n = 55 to 140, are extrapolated as a function of the geometric dependence of the cluster reduction energy to infinite size. These measurements make it possible to directly relate absolute reduction energies of these gaseous nanodrops containing Eu 3+ to the absolute reduction enthalpy of this ion in bulk solution. From this value, an absolute SHE potential of +4.11 V and a real proton solvation energy of −269.0 kcal/mol are obtained. The infrared photodissociation spectrum of Eu(H 2 O) 119-124 3+ indicates that the structure of the surface of the nanodrops is similar to that at the bulk air-water interface and that the hydrogen bonding of interior water molecules is similar to that in aqueous solution. These results suggest that the environment of Eu 3+ in these nanodrops and the surface potential of the nandrops are comparable to those of the condensed phase. This method for obtaining absolute potentials of redox couples has the advantage that no explicit solvation model is required, which eliminates uncertainties associated with these models, making this method potentially more accurate than previous methods.

Electrospray ionization (ESI) can be used to form multiply charged ions, which is beneficial for obtaining accurate molecular weight values and sequence information for proteins in many types of mass spectrometry (MS) measurements. The effects of surface tension, dipole moment, and dielectric constant (properties that have been implicated in the enhancement of protein charging in ESI) of 14 solution additives on the protein charge-state distributions that are formed in ESI for three proteins (ubiquitin, cytochrome c, and carbonic anhydrase II) were investigated. We discovered that two solution additives, propylene carbonate and ethylene carbonate, which have high surface tension and dipole moment values, can be used to form significantly higher protonation states of these three common test proteins than have been reported previously by use of other methods and additives, such as that obtained with benchmark "supercharging" additives (m-nitrobenzyl alcohol and sulfolane). By use of ethylene carbonate and propylene carbonate, nearly the entire charge state distributions of protonated ubiquitin and cytochrome c ions can be shifted to higher charge states than the theoretical maximum protein charging protonation limit in ESI that is predicted on the basis of proton-transfer reactivity. Ethylene carbonate, propylene carbonate, o-nitroanisole, m-nitrobenzyl alcohol, and sulfolane are all effective at increasing the extent of charging of deprotonated protein ions in negative ionization mode ESI-MS experiments. This indicates that physicochemical properties that are independent of polarity (e.g., surface tension) can be responsible for supercharging and should not be excluded without additional information. In addition, these data indicate that nonvolatile additives with high dipole moments are not necessarily sufficient to supercharge proteins. Overall, these results suggest that it should be possible to discover new additives that increase protein charging even further.

Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, β-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model.

Cytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O2 from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H2O2). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.