Insertion Intermediates Research Articles

The Conserved Third Transmembrane Segment of YidC Contacts Nascent Escherichia coli Inner Membrane Proteins

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.

Gas-phase activation of methane by ligated transition-metal cations.

Motivated by the search for ways of a more efficient usage of the large, unexploited resources of methane, recent progress in the gas-phase activation of methane by ligated transition-metal ions is discussed. Mass spectrometric experiments demonstrate that the ligands can crucially influence both reactivity and selectivity of transition-metal cations in bond-activation processes, and the most reactive species derive from combinations of transition metals with the electronegative elements fluorine, oxygen, and chlorine. Furthermore, the collected knowledge about intramolecular kinetic isotope effects associated with the activation of C-H(D) bonds of methane can be used to distinguish the nature of the bond activation as a mere hydrogen-abstraction, a metal-assisted mechanism or more complex reactions such as formation of insertion intermediates or sigma-bond metathesis.

Elimination of HBr from CH 3Br by Cu + and Au +: A DFT study

The elimination of HBr from CH 3Br by ground state of Cu + and Au + has been investigated by using DFT methods. A mechanism of one-step HBr elimination leading to CuCH 2 + was identified. For the formation of AuCH 2 +, besides the one-step mechanism, another two-step HBr elimination pathway through a C–Br bond insertion intermediate was also found, and this pathway is energetically more favorable than the one-step mechanism. The calculated reaction energy barriers show that the formation of AuCH 2 + is energetically much more favorable than the formation of CuCH 2 +. The experimental observations are well explained.

Insertion intermediate of annexin B12 is prone to aggregation on membrane interfaces

Insertion intermediate of annexin B12 is prone to aggregation on membrane interfaces

Theoretical study of the gas-phase ethane C–H and C–C bonds activation by bare niobium cation

The potential energy surfaces for the reaction of bare niobium cation with ethane, as a prototype of the C–H and C–C bonds activation in alkanes by transition metal cations, have been investigated employing the Density Functional Theory in its B3LYP formulation. All the minima and key transition states have been examined along both high- and low-spin surfaces. For both the C–H and C–C activation pathways the rate determining step is that corresponding to the insertion of the Nb cation into C–H and C–C bond, respectively. However, along the C–H activation reaction coordinate the barrier that is necessary to overcome is 0.13 eV below the energy of the ground state reactants asymptote, while in the C–C activation branch the corresponding barrier is about 0.58 eV above the energy of reactants in their ground state. The overall calculated reaction exothermicities are comparable. Since the spin of the ground state reactants is different from that of both H–Nb+–C2H5 and CH3–Nb+–CH3 insertion intermediates and products, spin multiplicity has to change along the reaction paths. All the obtained results, including Nb+–R binding energies for R fragments relevant to the examined PESs, have been compared with existing experimental and theoretical data.

Phosphinito- and phosphonito-oxazoline Pd(ii) complexes as CO/ethylene insertion intermediates: synthesis and structural characterization

The phosphinito-oxazoline ligand 4,4-dimethyl-2-[methoxy(diphenylphosphine)]-4,5-dihydrooxazole (2a) and the phosphonite-oxazoline ligand 4,4-dimethyl-2-[methoxy(6H-dibenz[c,e][1,2]oxaphosphorin)]-4,5-dihydrooxazole (8a) were prepared by deprotonation of (4,5-dihydro-4,4-dimethyloxazol-2-yl)methanol (1a) and reaction with the corresponding P-Cl function, similar to the ligands 2b (4,4-dimethyl-2-[1-oxy(diphenylphosphine)-1-methylethyl]-4,5-dihydrooxazole) and 8b (4,4-dimethyl-2-[1-oxy(6H-dibenz[c,e][1,2]oxaphosphorin)-1-methylethyl]-4,5-dihydrooxazole) reported previously. These ligands react with [PdClX(COD)] to give complexes of the type [PdClX(P,N)] (3a P,N = 2a, X = Cl; 4a P,N = 2a, X = Me; 4b P,N = 2b , X = Me; 9a P,N = 8a, X = Cl; 9b P,N = 8b, X = Cl; 10a P,N = 8a, X = Me; 10b P,N = 8b, X = Me). Complexes 4a,b and 10a,b reacted with AgCF(3)SO(3) to yield [PdMe(P,N)OSO(2)CF(3)] 5a,b and 11a,b, respectively. From the stepwise insertion reaction of CO and ethylene into the Pd-C bond of 5a and 11a,b, the alkyl ketone chelate complexes [Pd{CH(2)CH(2)C(O)Me}(P,N)]CF(3)SO(3) 7a and 14a,b respectively, have been isolated and spectroscopically characterized. Complexes 3a.CH(2)Cl(2), 5a, 9b, 10a,b, [PdMe(H(2)O)(P,N)]CF(3)SO(3) 12b, (P,N = 8b) and 14a,b have also been characterized by X-ray crystallography and the structures of 14a,b represent still rare examples of structurally characterized CO/ethylene coupling products.

Energetic and topological analyses of the oxidation reaction between Mon (n = 1, 2) and N2O

The interaction between molybdenum, atom, and dimer, with nitrous oxide has been investigated using density functional theory. The analysis of the potential energy surfaces for both reactions has revealed that a single molybdenum atom can activate the N--O bond of N2O requiring a small activation energy. However, the presence of several intersystem crossings between three different spin states, namely, septet, quintet and triplet states, seems to be the major constraint to the Mo + N2O reaction. Contrarily, the low-lying excited states (triplet and quintet) do not participate in the reaction between the molybdenum dimer and N2O. The latter reaction fully evolves on the singlet spin surface. Three different regions have been distinguished along the pathway: formation of an adduct complex, formation of an inserted compound, and the N2 detachment. The connection between the two first regions has been characterized by the formation of a special complex in which the N--O bond is so weakened that it could be considered as a first step in the insertion process. It has been shown that the topological changes along the pathways provide a clear explanation for the geometrical changes that occur along the reaction pathway. In summary, the detachment of the N2 molecule is found to be kinetically an effective process for both reactions, owing to the high exothermicity and consequently to the high internal energy of the insertion intermediates. However, in the case of Mo atom, the reaction should be a slow process due to the presence of spin-forbidden transitions. These results fully agree with previous experimental works.

DFT study on the reaction of neutral Ti and Ni atoms with CS 2

The reaction mechanisms of the ground state ( 3F) of neutral Ti atom, the lowest triplet ( 3F) and singlet state ( 1D) of Ni atom with CS 2: M+CS 2→MS+CS (M=Ti, Ni) have been investigated by using DFT method B3LYP with DZVP basis set. Two reaction channels have been identified for the reactions of both Ti ( 3F) and Ni ( 3F) atoms with CS 2, and one channel has been identified for the reaction of Ni ( 1D) with CS 2. These reactions are initiated by different mechanisms, but all the reaction channels involve the insertion of metal atoms into the C–S bond of CS 2 yielding insertion intermediates SMCS from C–S bridged intermediates M–(η 2-CS)S or cyclic complexes M–(η 2-S 2)C. The reaction of triplet ground state Ti with CS 2 requires no activation energy, whereas the reactions of Ni atom with CS 2 proceed with 7, 20 and 17 kcal/mol activation energies for the two triplet and one singlet pathways, respectively. The reaction of Ni with CS 2 was shown to occur preferentially on triplet surface and the experimentally observed species, Ni–(η 2-CS)S and SNiCS, have been explained according to the mechanisms revealed in this work.

Reactions of Cu+(1S,3D) and Au+(1S,3D) with CH3Br

The reactions of Cu+(1S,3D) and Au+(1S,3D) with CH3Br have been carried out in the gas phase at 150 K using a drift cell. State specific product channels were identified by observing the occurrence of product ions when ionization conditions were manipulated to either produce or eliminate excited states of the two metal ions. Reactant ion state distributions were determined by electronic state chromatography. State-specificity was confirmed by correlating reactant and product arrival time distributions. Association products correlate to singlet metal ion states for both Cu+ and Au+. Additionally, Cu+(3D) participates in association under these conditions. The 3D state of both metals exhibits Br abstraction, and CH3 is abstracted by Au+(3D). Formation of AuCH2+ exclusively via Au+(1S) is also indicated. State-specificity with respect to bimolecular channels for both metals is consistent with thermochemical requirements and formal conservation of electron spin. Parallel formation of AuCH2+ and Au+·CH3Br suggests a mechanism leading to HBr elimination that involves a long-lived insertion intermediate.

Mechanism of vinylic and allylic carbon-fluorine bond activation of non-perfluorinated olefins using Cp*(2)ZrH(2).

Cp(2)ZrH(2) (1) (Cp = pentamethylcyclopentadienyl) reacts with vinylic carbon-fluorine bonds of CF(2)=CH(2) and 1,1-difluoromethylenecyclohexane (CF(2)=C(6)H(10)) to afford Cp(2)ZrHF (2) and hydrodefluorinated products. Experimental evidence suggests that an insertion/beta-fluoride elimination mechanism is occurring. Complex 1 reacts with allylic C-F bonds of the olefins, CH(2)=CHCF(3), CH(2)=CHCF(2)CF(2)CF(2)CF(3), and CH(2)=C(CF(3))(2) to give preferentially 2 and CH(3)-CH=CF(2), CH(3)-CH=CF-CF(2)CF(2)CF(3), and CF(2)=C(CF(3))(CH(3)), respectively, by insertion/beta-fluoride elimination. In the reactions of 1 with CH(2)=CHCF(3) and CH(2)=CHCF(2)CF(2)CF(2)CF(3), both primary and secondary alkylzirconium olefin insertion intermediates were observed in the (1)H and (19)F NMR spectra at low temperature. A deuterium labeling study revealed that more than one olefin-dihydride complex is likely to exist prior to olefin insertion. In the presence of excess 1 and H(2), CH(2)=CHCF(3) and CH(2)=CHCF(2)CF(2)CF(2)CF(3) are reduced to propane and (E)-CH(3)CH(2)CF=CFCF(2)CF(3), respectively.

Theoretical Study of the Reaction of Yttrium with Formaldehyde

This work describes single-(DFT, CCSD(T)) and multireference (CASSCF, CASPT2) theoretical calculations on the reaction of yttrium atoms with formaldehyde studied recently in crossed molecular beam experiments. The reaction is shown to proceed through the exothermic formation of a side-bound π-complex followed by C-H insertions which branch out to competing pathways to products. Dihydridoyttrium(II) is formed through the decomposition of the double insertion product, whereas carbonylyttrium(0) and high-energy formylyttrium-(I) result from the single insertion intermediate. The product and transition state energetics are consistent with experimental results and allow one to rule out the direct reductive elimination pathway for the formation of carbonylyttrium(0).

YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids.

Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids.

Methane to Methanethiol Conversion by FeS+. A Combined Experimental and Theoretical Study†

The reaction of FeS+ with methane is examined by guided ion beam mass spectrometry and density functional theory employing the B3LYP/6-311+G* level of theory. For the FeS+/CD4 system examined in the experiments, two major product ions, Fe+ and FeSD+, are observed along with minor channels leading to FeCD3+, FeSCD3+, and FeSCD+. All products are formed in endothermic processes. The measured thresholds for the formations of Fe+ and FeSD+ are compared with computational data as well as literature thermochemistry. In the theoretical approach, two competing reaction mechanisms for the formation of Fe+, concomitant with neutral methanethiol, are investigated and used to interpret the experimental data. The lowest-energy path involves a formal 1,2-addition of H3C−H across the Fe+−S bond to generate a CH3FeSH+ insertion intermediate. This bond activation step involves spin inversion from the sextet to the quartet surface en route to the products. The occurrence of the second conceivable pathway resulting in formation of HFeSCH3+ as an intermediate can be ruled out because of the high-energy demand associated with overcoming the insertion barrier along this pathway.

New Imine−Phosphine Palladium Complexes Catalyze Copolymerization of CO−Ethylene and CO−Norbornylene and Provide Well-Characterized Stepwise Insertion Intermediates of Various Unsaturated Substrates

New imine−phosphine palladium complexes [Pd(P−N)(CH3)(CH3CN)](BF4) (P−N = o-(diphenylphosphino)-N-benzaldimine derivatives) are synthesized and are found to be active for copolymerization of CO−ethylene and CO−norbornylene, respectively. Stepwise addition of carbon monoxide and various olefins into the new complexes is investigated.

Ab initio study on the reaction of Sc+ + CH4 ? Sc+(SINGLE BOND)CH2 + H2

An ab initio study on the reaction of the ground state (3D) and the excited state (1D) of Sc+ with methane was performed. Reaction channels on the singlet and triplet potential surface (PES) and the reaction mechanism are examined and discussed. Three regions of the potential surface was studied: the molecular complex, the C(SINGLE BOND)H insertion products, and the transition states for the reaction. Comparisons between singlet and triplet PESs show that the excited state (1D) of Sc+ has more reactivity with methane than does the ground state (3D) due to the spin quantum number conservation with the more stable insertion intermediate. © 1997 John Wiley & Sons, Inc.

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.

Conversion of CH4 to CH3OH: Reactions of CoO+ with CH4 and D2, Co+ with CH3OD and D2O, and Co+(CH3OD) with Xe

The mechanisms and energetics involved in the conversion of CH{sub 4} to CH{sub 3}OH by CoO{sup +} are examined by using guided ion beam mass spectrometry. The forward and reverse reactions, CO{sup +} + CH{sub 4} CO{sup +} + Ch{sub 3}OH, the collisional activation of Co{sup +}(CH{sub 3}OH), and the related reactions, CoO{sup +} + D{sub 2} Co{sup +} + D{sub 2}O, are studied. It is found that the oxidations of methane and D{sub 2} by CoO{sup +}, both exothermic reactions, do not occur until overcoming activation barriers of 0.56 x 0.08 and 0.75 x 0.04 eV, respectively. The behavior of the forward and reverse reactions in both systems is consistent with reactions that proceed via the insertion intermediates R-Co{sup +}-OH, where R = CH{sub 3} or H. The barrier is probably attributable to a four-centered transition state associated with addition of RH across the CoO{sup +} bond. In the Co{sup +} + CH{sub 3}OH system (where CH{sub 3}OD labeled reactant is used), reactions explained by initial C-H and O-H activation are also observed. The reaction mechanisms and potential energy surfaces for these systems are derived and discussed. Phase space theory calculations are used to help verify these details formore » the CoO{sup +} + D{sub 2} system. Thermochemistry for several species including CoOH{sup +}, CoD{sup +}, CoH, CoCH{sub 3}{sup +}, Co{sup +}(CH{sub 3}OD), CoOCH{sub 3}{sup +}, and possibly OCoCH{sub 3}{sup +} is derived from measurements of reaction thresholds. 59 refs., 8 figs., 4 tabs.« less

The role of electrostatic charge in the membrane insertion of colicin A. Calculation and mutation.

The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.

Dynamics of the reactions of Zn(4s4p 3P1) with H2, HD, and D2: Rotational and vibrational quantum-state distributions of ZnH (ZnD) products

The complete initial vibrational and rotational quantum state distributions of ZnH(ZnD) products in the reactions of Zn(4s4p 3P1) with H2, HD, and D2 have been determined using a laser ‘‘pump-and-probe’’ technique. The most striking result is that the quantum-state distributions of ZnH (or ZnD) products are essentially unchanged when the mass of the leaving atom is doubled, from H to D. It is suggested that this indicates that simple impulsive bond breaking cannot play a large role in the reaction of Zn(3P1) with H2, and that potential surface anisotropy in the decomposition of bent H–Zn–H insertion intermediates could be responsible for the rotational energy distributions of the products. Similar isotopic results for reactions of Cd(5s5p 3P1), Hg(6s6p 3P1), and O(1D2) with H2, HD, and D2 are noted, and the general implications of the lack of an isotope effect are discussed in detail. The branching ratio of ZnD vs ZnH formation in the reaction of Zn(3P1) with HD was determined to be 1.1±0.2 and it was pointed out that several ‘‘insertion’’ reactions have now been shown to have branching ratios for reaction with HD which are very near 1.0, inconsistent with earlier qualitative arguments that such processes should lead to high branching ratios.

Initial internal energy distributions of CdH (CdD) produced in the reaction of Cd(5s5p 3P 1) with H 2, D 2, and HD

Initial rotational quantum-state distributions of CdH (CdD) formed in the endoergic reactions of Cd(5s5p 3P 1) with H 2, HD, and D 2 were determined for reactants under thermal conditions at 325 K. Unusual isotope effects were observed, very similar to those reported for the analogous exoergic reactions of Zn(4s4p 3P 1). The results were successfully rationalized by a simple transition-state model in which small, “late” activation barriers for the decomposition of H—Cd—H, H—Cd—D, and D—Cd—D insertion intermediates were postulated.