Conservative and Atypical Ferritins of Sponges.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 ß subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.

Detergent-free mass spectrometry of membrane protein complexes.

We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.

Dual stoichiometry and subunit organization in the ClpP1/P2 protease from the cyanobacterium Synechococcus elongatus.

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. To investigate the proteolytic core of the ClpXP1/P2 protease from the cyanobacterium Synechococcus elongatus we have used a non-denaturing mass spectrometry approach. We show that the proteolytic core is a double ring tetradecamer consisting of an equal number of ClpP1 and ClpP2 subunits with masses of 21.70 and 23.44 kDa, respectively. Two stoichiometries are revealed for the heptameric rings: 4ClpP1+3ClpP2 and 3ClpP1+4ClpP2. When combined in the double ring the stoichiometries are (4ClpP1+3ClpP2)+(3ClpP1+4ClpP2) and 2×(3ClpP1+4ClpP2) with a low population of a 2×(4ClpP1+3ClpP2) tetradecamer. The assignment of the stoichiometries is confirmed by collision-induced dissociation of selected charge states of the intact heptamer and tetradecamer. Presence of the heterodimers, heterotetramers and heterohexamers, and absence of the mono-oligomers, in the mass spectra of the partially denatured protease indicates that the ring complex consists of a chain of ClpP1/ClpP2 heterodimers with the ring completed by an additional ClpP1 or ClpP2 subunit.

Mass-selective soft-landing of protein assemblies with controlled landing energies.

Selection and soft-landing of bionanoparticles in vacuum is potentially a preparative approach to separate heterogeneous mixtures for high-resolution structural study or to deposit homogeneous materials for nanotechnological applications. Soft-landing of intact protein assemblies however remains challenging, due to the difficulties of manipulating these heavy species in mass-selective devices and retaining their structure during the experiment. We have developed a tandem mass spectrometer with the capability for controlled ion soft-landing and ex situ visualization of the soft-landed particles by means of transmission electron microscopy. The deposition conditions can be controlled by adjusting the kinetic energies of the ions by applying accelerating or decelerating voltages to a set of ion-steering optics. To validate this approach, we have examined two cage-like protein complexes, GroEL and ferritin, and studied the effect of soft-landing conditions on the method's throughput and the preservation of protein structure. Separation, based on mass-to-charge ratio, of holo- and apo-ferritin complexes after electrospray ionization enabled us to soft-land independently the separated complexes on a grid suitable for downstream transmission electron microscopy analysis. Following negative staining, images of the soft-landed complexes reveal that their structural integrity is largely conserved, with the characteristic central cavity of apoferritin, and iron core of holoferritin, surviving the phase transition from liquid to gas, soft-landing, and dehydration in vacuum.

Changes in the geometries of C2H2 and C2H4 on coordination to CuCl revealed by broadband rotational spectroscopy and ab-initio calculations.

The molecular geometries of isolated complexes in which a single molecule of C2H4 or C2H2 is bound to CuCl have been determined through pure rotational spectroscopy and ab-initio calculations. The C2H2···CuCl and C2H4···CuCl complexes are generated through laser vaporization of a copper rod in the presence of a gas sample undergoing supersonic expansion and containing C2H2 (or C2H4), CCl4, and Ar. Results are presented for five isotopologues of C2H2···CuCl and six isotopologues of C2H4···CuCl. Both of these complexes adopt C(2v), T-shaped geometries in which the hydrocarbon binds to the copper atom through its π electrons such that the metal is equidistant from all H atoms. The linear and planar geometries of free C2H2 and C2H4, respectively, are observed to distort significantly on attachment to the CuCl unit, and the various changes are quantified. The ∠(*-C-H) parameter in C2H2 (where * indicates the midpoint of the C≡C bond) is measured to be 192.4(7)° in the r0 geometry of the complex representing a significant change from the linear geometry of the free molecule. This distortion of the linear geometry of C2H2 involves the hydrogen atoms moving away from the copper atom within the complex. Ab-initio calculations at the CCSD(T)(F12*)/AVTZ level predict a dihedral ∠(HCCCu) angle of 96.05° in C2H4···CuCl, and the experimental results are consistent with such a distortion from planarity. The bonds connecting the carbon atoms within each of C2H2 and C2H4, respectively, extend by 0.027 and 0.029 Å relative to the bond lengths in the isolated molecules. Force constants, k(σ), and nuclear quadrupole coupling constants, χ(aa)(Cu), [χ(bb)(Cu) - χ(cc)(Cu)], χ(aa)(Cl), and [χ(bb)(Cl) - χ(cc)(Cl)], are independently determined for all isotopologues of C2H2···CuCl studied and for four isotopologues of C2H4···CuCl.

The kinetics and product state distributions from gas-phase reactions of small atomic and molecular cations with C2H4, C2H3F, 1,1-C2H2F2, C2HF3 and C2F4.

The reactions of twenty one gas-phase cations with C2H3F, 1,1-C2H2F2, C2HF3 and C2F4 have been studied in a selected ion flow tube at 298 K. The cations are both atomic and molecular with recombination energies in the range 6-22 eV, and the kinetics and branching ratios into product ions are revealed for all the reactions. These data, together with that from an earlier study of reactions of C(x)F(y)(+) with these four fluorinated ethenes (J. Phys. Chem. A., 2012, 116, 8119), are compared with the reactions of these ions with C2H4, where available. Nearly all the reactions have a rate coefficient close to the collisional value calculated by either Langevin or modified average dipole orientation theories. The products of the reactions of N(+) and N2(+) with C2H4 are found to be anomalous, compared to their reactions with the four fluorinated ethenes. The branching ratios into product cations are compared with those from a high resolution (ca. 0.002 eV) photoionisation (hν = 10-22 eV) study of C2H3F, 1,1-C2H2F2, C2HF3 and C2F4 (Phys. Chem. Chem. Phys., 2012, 14, 3935) in order to gauge the importance of electron transfer in ion-molecule reactions. The higher the recombination energy of the cation, the better the agreement between the two sets of product branching ratios. Where there is disagreement at lower recombination energies, it appears that there is more fragmentation of the products in the photoionisation experiment compared to the ion-molecule reactions.

Differentiating Inhibition Selectivity and Binding Affinity of Isocitrate Dehydrogenase 1 Variant Inhibitors.

Isocitrate dehydrogenase (IDH) 1/2 gain-of-function variants catalyze the production of the oncometabolite 2-hydroxyglutarate and are validated targets for leukemia treatment. We report binding and inhibition studies on 13 IDH1/2 variant inhibitors, including clinical candidates and drugs, with wild-type (wt) IDH1 and its cancer-associated variant, IDH1 R132H. Interestingly, all the variant inhibitors bind wt IDH1 despite not, or only weakly, inhibiting it. Selective inhibition of the IDH1 R132H variant over wt IDH1 does not principally relate to the affinities of the inhibitors for the resting forms of the enzymes. Rather, the independent binding of Mg2+ and 2-oxoglutarate to the IDH1 variant makes the variant more susceptible to allosteric inhibition, compared to the tighter binding of the isocitrate-Mg2+ complex substrate to wt IDH1. The results highlight that binding affinity need not correlate with inhibition selectivity and have implications for interpretation of inhibitor screening results with IDH and related enzymes using turnover versus binding assays.

A prototype transition-metal olefin complex C2H4···AgCl synthesised by laser ablation and characterised by rotational spectroscopy and ab initio methods.

C(2)H(4)···Ag-Cl has been synthesised in the gas phase in a pulsed-jet, Fourier-transform microwave spectrometer by the reaction of laser-ablated metallic silver with carbon tetrachloride to give AgCl, which subsequently reacts with ethene to give the complex. The ground-state rotational spectra of six isotopologues (C(2)H(4)···(107)Ag(35)Cl, C(2)H(4)···(109)Ag(35)Cl, C(2)H(4)···(107)Ag(37)Cl, C(2)H(4)···(109)Ag(37)Cl, (13)C(2)H(4)···(107)Ag(35)Cl, and (13)C(2)H(4)···(109)Ag(35)Cl) were recorded and analysed to give rotational constants A(0), B(0), and C(0), centrifugal distortion constants Δ(J) and Δ(JK), and Cl nuclear quadrupole coupling constants χ(aa)(Cl) and χ(bb)(Cl)-χ(cc)(Cl). These spectroscopic constants were interpreted in terms of a geometry for C(2)H(4)···Ag-Cl of C(2V) symmetry in which the AgCl molecule lies along the C(2) axis of ethene that is perpendicular to the C(2)H(4) plane. The Ag atom forms a bond to the midpoint (*) of the ethene π bond. A partial r(s)-geometry and a r(0)-geometry were determined, with the values r(*···Ag) = 2.1719(9) Å, r(C-C) = 1.3518(4) Å, and r(Ag-Cl) = 2.2724(8) Å obtained in the latter case. The C-C bond lengthens on formation of the complex. Detailed ab initio calculations carried out at the CCSD(T)/cc-pVQZ level of theory give results in good agreement with experiment and also reveal that the ethene molecule undergoes a small angular distortion. The distortion is such that the four H atoms move in a direction away from Ag but remain coplanar. The two C atoms are no longer contained in this plane, however. The electric charge redistribution when C(2)H(4)···Ag-Cl is formed and the strength of the π···Ag bond are discussed.

Monohydrates of cuprous chloride and argentous chloride: H2O⋠⋠⋠CuCl and H2O⋠⋠⋠AgCl characterized by rotational spectroscopy and ab initio calculations.

Pure rotational spectra of the ground vibrational states of ten isotopologues of each of H(2)O⋅⋅⋅CuCl and H(2)O⋅⋅⋅AgCl have been measured and analyzed to determine rotational constants and hyperfine coupling constants for each molecule. The molecular structure and spectroscopic parameters determined from the experimental data are presented alongside the results of calculations at the CCSD(T) level. Both experiment and theory are consistent with structures that are nonplanar at equilibrium. The heavy atoms are collinear while the local C(2) axis of the water molecule intersects the axis defined by the heavy atoms at an angle, φ = 40.9(13)° for Cu and φ = 37.4(16)° for Ag. In the zero-point state, each molecule is effectively planar, undergoing rapid inversion between two equivalent structures where φ has equal magnitude but opposite sign. The equilibrium geometry has C(s) symmetry, however. The ab initio calculations confirm that the timescale of this inversion is at least an order of magnitude faster than that of rotation of the molecule in the lowest rotational energy levels. The molecular geometries are rationalized using simple rules that invoke the electrostatic interactions within the complexes. Centrifugal distortion constants, Δ(J) and Δ(JK), nuclear quadrupole coupling constants, χ(aa)(Cu), χ(aa)(Cl), (χ(bb) - χ(cc))(Cu), and (χ(bb) - χ(cc))(Cl), and the nuclear spin-rotation constant of the copper atom, C(bb)(Cu)+C(cc)(Cu), are also presented.

Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1.

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a 'gain-of-function' to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

Influence of the quick-to-court gene deletion on courtship behaviour of Drosophila melanogaster.

Using an original method, we have received Drosophila melanogaster with a deficiency including a complete sequence of quick-to-court gene. In this report, we describe the behavioural features of this new deletion mutant. There were no serious deviations from the normal mating behaviour in flies with the deletion, but the behaviour of deletion mutants still had some features. Of all the elements, only the frequency of licking significantly increased in mutants. The duration of mating elements did not change in flies with deletion, and the latent period decreased only for following the female and licking. We have found that mutant males produce more courtship song than control males when courting Oregon R females as estimated by the pulse song index. In our experiment, mutant females provoked much less pulse song production by Oregon R males than control females do. Moreover, Oregon R males initiate courtship song towards mutant females later than towards control females. In other words, the study of pulse song production showed that the deficiency in females leads to a decrease in the intensity of courtship of wild-type males, whereas the deficiency in males leads to more intensive care for wild-type females.

Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding.

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective 'peptibitors' inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.

Top-down mass analysis of protein tyrosine nitration: comparison of electron capture dissociation with "slow-heating" tandem mass spectrometry methods.

Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the "slow-heating" threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the "slow-heating" methods.

Iron metabolic pathways in the processes of sponge plasticity.

The ability to regulate oxygen consumption evolved in ancestral animals and is intrinsically linked to iron metabolism. The iron pathways have been intensively studied in mammals, whereas data on distant invertebrates are limited. Sea sponges represent the oldest animal phylum and have unique structural plasticity and capacity to reaggregate after complete dissociation. We studied iron metabolic factors and their expression during reaggregation in the White Sea cold-water sponges Halichondria panicea and Halisarca dujardini. De novo transcriptomes were assembled using RNA-Seq data, and evolutionary trends were analyzed with bioinformatic tools. Differential expression during reaggregation was studied for H. dujardini. Enzymes of the heme biosynthesis pathway and transport globins, neuroglobin (NGB) and androglobin (ADGB), were identified in sponges. The globins mutate at higher evolutionary rates than the heme synthesis enzymes. Highly conserved iron-regulatory protein 1 (IRP1) presumably interacts with the iron-responsive elements (IREs) found in mRNAs of ferritin (FTH1) and a putative transferrin receptor NAALAD2. The reaggregation process is accompanied by increased expression of IRP1, the antiapoptotic factor BCL2, the inflammation factor NFκB (p65), FTH1 and NGB, as well as by an increase in mitochondrial density. Our data indicate a complex mechanism of iron regulation in sponge structural plasticity and help to better understand general mechanisms of morphogenetic processes in multicellular species.

Correction: Iron metabolic pathways in the processes of sponge plasticity.

[This corrects the article DOI: 10.1371/journal.pone.0228722.].

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda.

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.

Selected ion flow tube study of the ion-molecule reactions of monochloroethene, trichloroethene, and tetrachloroethene.

Data for the rate coefficients and product cations of the reactions of a large number of atomic and small molecular cations with monochloroethene, trichloroethene, and tetrachloroethene in a selected ion flow tube at 298 K are reported. The recombination energy of the ions range from 6.27 (H3O(+)) through to 21.56 (Ne(+)) eV. Collisional rate coefficients are calculated by modified average dipole orientation theory and compared with experimental values. Thermochemistry and mass balance predict the most feasible neutral products. Together with previously reported results for the three isomers of dichloroethene ( Mikhailov, V. A. ; Parkes, M. A. ; Tuckett, R. P. ; Mayhew, C. A. J. Phys. Chem. A 2006, 110, 5760 ), the fragment ion branching ratios have been compared with those from threshold photoelectron photoion coincidence spectroscopy over the photon energy range of 9-22 eV to determine the importance or otherwise of long-range charge transfer. For ions with recombination energy in excess of the ionization energy of the chloroethene, charge transfer is energetically allowed. The similarity of the branching ratios from the two experiments suggest that long-range charge transfer is dominant. For ions with recombination energy less than the ionization energy, charge transfer is not allowed; chemical reaction can only occur following formation of an ion-molecule complex, where steric effects are more significant. The products that are now formed and their percentage yields are a complex interplay between the number and position of the chlorine atoms with respect to the C=C bond, where inductive and conjugation effects can be important.