Importance of iron as the metal ion in peptide deformylase: a biomimetic computational study

Peptide deformylase (PDF), a metalloamidase which catalyzes a deformylation step during eubacterial protein biosynthesis, shows a peculiar preference for Fe^II as its active site metal ion (in particular, as opposed to Zn^II, which is far more common among this class of enzymes). In order to explore the origin of this preference, density functional theory (DFT) calculations have been carried out using a biomimetic heteroscorpionate N₂S_thiolate ligand system (L) and the metal centers Fe^II, Zn^II, and Co^II. Comparison of computed ML(formate) complexes to crystal structures of PDF?Cformate complexes illustrates the viability of the biomimetic ligand for investigating the PDF chemistry. pK_a calculations on [ML(H₂O)]⁺ complexes show that the metal centers are effective Lewis acids in activating the water molecule to allow formation of a nucleophilic hydroxide ligand. Computed oxidation potentials predict the ML(OH) and ML(formate) complexes not to be unstable with respect to oxidation. However, while each of the metal centers was therefore seen to be suitable for PDF chemistry, examination of the entire deformylation reaction showed Fe^II to be uniquely suited to PDF. The deformylation reaction was thermodynamically and kinetically optimal with Fe^II as the metal center. This is attributed to the charge transfer that occurs from the thiolate ligand to the Fe^II center during the reaction and to the relative coordinative flexibility of Fe^II that allows for facile interconversion between tetra- and pentacoordination, leading to greater activation of the substrate carbonyl at the nucleophilic attack transition state.     

Theoretical study of the catalytic mechanism and metal-ion dependence of peptide deformylase

The reaction pathway of deformylation catalyzed by E. coli peptide deformylase (PDF) has been investigated by the density functional theory method of PBE1PBE on a small model and by a two-layer ONIOM method on a realistic protein model. The deformylation proceeds in sequential steps involving nucleophilic addition of metal-coordinated water/hydroxide to the carbonyl carbon of the formyl group, proton transfer, and cleavage of the C-N bond. The first step is rate-determining for the deformylation, which occurs through a pentacoordinated metal center. The estimated activation energies with the ONIOM method are about 23.0, 15.0, and 14.9 kcal/mol for Zn-, Ni-, and Fe-PDFs, respectively. These calculated barriers are in close agreement with experimental observations. Our results demonstrate that the preference for metal coordination geometry exerts a significant influence on the catalytic activity of PDFs by affecting the activation of the carbonyl group of the substrate, the deprotonation of the metal-coordinated water, and the stabilization of the transition state. This preference for coordination geometry is mainly determined by the ligand environment and the intrinsic electronic structures of the metal center in the active site of the PDFs.     

Ag+ Selective Macrocycles Containing Soft Ligating Moieties and Regulation of Ag+ Binding

Strategies are discussed for the design of Ag+ selective macrocyclic molecules, together with the structures of the Ag+ complexes. One of the most useful and basic methods is to incorporate heteroatoms, such as nitrogens and sulfurs, and heterocycles into the macrocyclic framework. A side arm containing the heteroatom also enhances Ag+ selectivity tremendously. A sulfide chain outside or inside the macroring contributes to highly selective Ag+ binding. Soft alkenyl and alkynyl carbons arranged in a macrocyclic fashion bind Ag+ preferentially. Regulation of Ag+ binding by redox reactions and by metal ligation is also described.     

Gas phase studies of the interactions of Fe2+ with cysteine-containing peptides

Gas-phase complexes of cysteine-containing peptides and Fe²⁺ were produced by fast atom bombardment and studied by tandem mass spectrometry. Specific and strong interactions of the iron and sulfur from the thiol group of the cysteine side chain are preserved in the gas phase and are the basis for highly specific fragmentation to give abundant [a_n − 2H + Fe]⁺ ions, where n is position of the cysteine residue from the N-terminus of peptide. Metal/peptide complexes containing more than one Cys residue were also investigated; they display similar chemistry upon collisionally activated decompositions, indicating that the Fe²⁺ ion primarily binds at cysteine sites.     

Homo- and heteropolynuclear Ni2+ and Cu2+ complexes of polytopic ligands, consisting of a tren unit substituted with three 12-membered tetraazamacrocycles

Two new polytopic ligands L1 and L2 have been synthesized. They consist of a central tren unit to which three 1,4,7,10-tetraazacyclododecane rings are attached via an ethylene and a trimethylene bridge, respectively. The complexation properties of L1 and L2 towards Cu(2+) and Ni(2+) were studied by potentiometric pH titration, UV-Vis, EPR spectroscopy and kinetic techniques. As a comparison, the Cu(2+) and Ni(2+) complexes with L3 (1-(N-methyl-2-aminoethyl-1,4,7,10-tetraazacyclododecane)) were also investigated. The crystal structures of [CuL3H(H(2)O)](ClO(4))(3) and [NiL3Cl](ClO(4)) were solved and show that the side chain in its protonated form is not involved in coordination, whereas deprotonated it binds to the metal ion. The thermodynamically stable 3:1 complexes of L1 or L2 have a metal ion in the three macrocyclic units. However, when three equivalents of Cu(2+) are added to L1 or L2 the metal ion first binds to the tren unit and only then to the macrocycles. The kinetics of the different steps of complexation have been studied and a mechanism is proposed.     

Formate dehydrogenase--a biocatalyst with novel applications in organic chemistry

In the field of industrial biocatalysis, formate dehydrogenase (FDH) is well established, in particular for its broad application in cofactor regeneration. Further applications have been limited by the enzyme's narrow range of substrates. These restrictions have been overcome now by the finding, that the enzyme is capable of selectively cleaving formic acid esters to the respective alcohol. Five homologous alkyl formates and phenyl formate as an aromatic ester were converted quantitatively by FDH from Candida boidinii in a batch reaction within 3 to 5 h. The substrates were turned irreversibly into carbon dioxide and the respective alcohol through hydride abstraction from the formyl group with full conversion. The mechanism shows parallels to hydrolysis reactions of the A(AC)1-type. K(M)-values and reactions rates of the tested formic acid esters display a tendency to higher conversion rates with increasing chain length. FDH emerged to be a superior deformylation catalyst compared to hydrolases as well as classical catalysts, as was shown by the selective deformylation of 1-acetoxy-4-formoxy butane (92%) and 1,3-bis(3-formoxypropyl)tetramethyldisiloxane. In particular its capability to distinguish between formic acid esters and non-formic acid esters renders the method particularly suitable for protective group chemistry. Furthermore the completeness of deformylation allows for converting substrates highly incompatible with aqueous media like siloxanes within a few hours.     

Pb2+ as modulator of protein-membrane interactions

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 ? resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane F?rster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.     

Proximal ligand electron donation and reactivity of the cytochrome P450 ferric-peroxo anion

CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compound I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addition of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Analysis of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compound I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pK(a) of the ferric-peroxo anion, which favors its protonation and, therefore, Compound I formation.     

Selective metal promoted hydrolysis of nitrile groups in the side chain of tetraazamacrocyclic Cu2+-complexes

The metal promoted hydrolysis of nitrile groups in the side chains of tetraazamacrocyclic Cu2+ complexes has been studied by stopped-flow techniques. It is shown that the reaction proceeds by an intramolecular attack of an axially coordinated OH- onto the nitrile group to give the corresponding amide. In alkaline solution the amide then deprotonates and binds to the axial position of the Cu2+ thus preventing further coordination of an OH-. This explains mechanistically that in the Cu2+ complexes of macrocycles carrying two nitrile functions only one is selectively hydrolysed. The nitrile hydrolysis has also been used on a preparative scale to synthesize tetraazamacrocycles with two different side chains. X-Ray diffractions of several products are presented to confirm the structures and the results from the kinetics and equilibria measurements.     

Infrared multiple photon dissociation spectroscopy of cationized histidine: effects of metal cation size on gas-phase conformation

The gas phase structures of cationized histidine (His), including complexes with Li(+), Na(+), K(+), Rb(+), and Cs(+), are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy utilizing light generated by a free electron laser, in conjunction with quantum chemical calculations. To identify the structures present in the experimental studies, measured IRMPD spectra are compared to spectra calculated at B3LYP/6-311+G(d,p) (Li(+), Na(+), and K(+) complexes) and B3LYP/HW*/6-311+G(d,p) (Rb(+) and Cs(+) complexes) levels of theory, where HW* indicates that the Hay-Wadt effective core potential with additional polarization functions was used on the metals. Single point energy calculations were carried out at the B3LYP, B3P86, and MP2(full) levels using the 6-311+G(2d,2p) basis set. On the basis of these experiments and calculations, the only conformation that reproduces the IRMPD action spectra for the complexes of the smaller alkali metal cations, Li(+)(His) and Na(+)(His), is a charge-solvated, tridentate structure where the metal cation binds to the backbone carbonyl oxygen, backbone amino nitrogen, and nitrogen atom of the imidazole side chain, [CO,N(α),N(1)], in agreement with the predicted ground states of these complexes. Spectra of the larger alkali metal cation complexes, K(+)(His), Rb(+)(His), and Cs(+)(His), have very similar spectral features that are considerably more complex than the IRMPD spectra of Li(+)(His) and Na(+)(His). For these complexes, the bidentate [CO,N(1)] conformer in which the metal cation binds to the backbone carbonyl oxygen and nitrogen atom of the imidazole side chain is a dominant contributor, although features associated with the tridentate [CO,N(α),N(1)] conformer remain, and those for the [COOH] conformer are also clearly present. Theoretical results for Rb(+)(His) and Cs(+)(His) indicate that both [CO,N(1)] and [COOH] conformers are low-energy structures, with different levels of theory predicting different ground conformers.     

Prins Cyclization Catalyzed by a FeIII/Trimethylsilyl Halide System: The Oxocarbenium Ion Pathway versus the [2+2] Cycloaddition

The different factors that control the alkene Prins cyclization catalyzed by iron(III) salts have been explored by means of a joint experimental–computational study. The iron(III) salt/trimethylsilyl halide system has proved to be an excellent promoter in the synthesis of crossed all‐cis disubstituted tetrahydropyrans, minimizing the formation of products derived from side‐chain exchange. In this iron(III)‐catalyzed Prins cyclization reaction between homoallylic alcohols and non‐activated alkenes, two mechanistic pathways can be envisaged, namely the classical oxocarbenium route and the alternative [2+2] cycloaddition‐based pathway. It is found that the [2+2] pathway is disfavored for those alcohols having non‐activated and non‐substituted alkenes. In these cases, the classical pathway, via the key oxocarbenium ion, is preferred. In addition, the final product distribution strongly depends upon the nature of the substituent adjacent to the hydroxy group in the homoallylic alcohol, which can favor or hamper a side 2‐oxonia‐Cope rearrangement.     

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization

High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in multiple charged ions. The most probable kinetic energy released (Tm.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b7" + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.     

Influences of peptide side chains on the metal ion binding site in metal ion-cationized peptides: Participation of aromatic rings in metal chelation

Aromatic side chains on amino acids influence the fragmentations of cationic complexes of doubly charged metal ions and singly deprotonated peptides. The metal ion interacts with an aromatic side chain and binds to adjacent amide nitrogens. When fragmentation occurs, this bonding leads to the formation of abundant metal-containing a-type ions by reactions that occur at the sites of amino acids that contain the aromatic side chain. Furthermore, formation of metal-containing immonium ions of the amino acids that contain the aromatic side chain also are formed. The abundant a-type ions may be useful in interpretation strategies in which it is necessary to locate in a peptide the position of an amino acid that bears an aromatic side chain.     

Structural elucidation of manganese(II), iron(III), cobalt(II), nickel(II), copper(II) and zinc(II) complexes of a new multidentate dihydrazino quinoxaline derivative

Summary Manganese(II), iron(III), cobalt(II), nickel(II), copper(II) and zinc(II) complexes of a new multidentate oxygen-nitrogen donor, bis(N-salicylidene)-2,3-dihydrazino-1,4-quinoxaline (H₂BSDHQ) were prepared and characterised by elemental analysis, conductance, thermal, spectral and magnetic data. H₂BSDHQ deprotonates to give a dibasic ONNO donor set in a trivalent iron(III) complex, which binds to the divalent metal ions in a bis-tridentate fashion, using two monobasic ONN donor sets, and resulting in polymeric complexes. Octahedral geometries are proposed for all these complexes, and preliminary studies show that they possess potential antimicrobial activity.     

Chiral electron deficient ruthenium helical coordination polymer as a catalyst for the epoxidation of substituted styrenes

An air and moisture stable ruthenium(Ⅲ) formate complex[Ru(HCO_2)Cl_2]_n has been synthesized and examined in the epoxidation of substituted styrenes.X-ray crystallographic data of this complex were determined and showed that the formate ligand coordinates to the ruthenium centers in a μ~2-η~2 fashion(syn,syn).Its asymmetric unit contains one Ru(Ⅲ) ion together with the half of a formate ligand and one chloride anion,which are bridged between the metal centers,forming a 1-D chain coordination polymer.This electron deficient helical coordination polymer was employed in the epoxidation of parafluorostyrene,affording the epoxide product in 92%yield.Natural chirality of this coordination polymer is applicable in asymmetric epoxidation reactions.     

First-row transition metal complexes of the strongly donating pentadentate ligand PY4Im

The new ligand PY4Im, which incorporates an axial N-heterocyclic carbene and four equatorial pyridine donors, is readily prepared on a multigram scale. Six-coordinate first row transition metal complexes of the general formula [(PY4Im)M(MeCN)](2+) (M = Fe, Co, Ni, Cu), where the PY4Im ligand coordinates in a square pyramidal pentadentate fashion, have been prepared. Structural, spectroscopic, and electrochemical characterization of these compounds provides evidence that PY4Im is a strongly donating ligand that favors the formation of low-spin complexes. Chemical oxidation of the iron(II) complex provides a low spin iron(III) complex, which has also been structurally and spectroscopically characterized. In the case of manganese(II), the PY4Im ligand is unable to either enforce a low-spin state or fully accommodate the metal ion. Rather, the ligand binds in a tridentate, face-capping mode.     

Catalytic mechanism and metal specificity of bacterial peptide deformylase: a density functional theory QM/MM study

Bacterial peptide deformylase (PDF) represents a novel class of mononuclear iron peptidase, and has an intriguing metal preference different from most other metalloproteases. Using a hybrid density functional theory (B3LYP) QM/MM method, we have theoretically investigated its catalytic mechanism and metal specificity by studying both Fe2+-PDF and Zn2+-PDF. In both forms of PDF, the conserved Glu133 residue is protonated in the reactant complex, and acts as a general acid during the reaction. The initial reaction step is the nucleophilic attack of the metal-bound hydroxide on the carbonyl carbon of the substrate. Our calculations indicate that the metal ion in Fe2+-PDF is always pentacoordinated during the reaction process, while that in Zn2+-PDF is only tetrahedrally coordinated and not bound to the substrate in the reactant complex. This difference in their metal coordination is suggested to account for the lower activity of Zn2+-PDF in comparison with Fe2+-PDF.     

Origins of the different metal preferences of Escherichia coli peptide deformylase and Bacillus thermoproteolyticus thermolysin: a comparative quantum mechanical/molecular mechanical study

The Escherichia coli peptide deformylase (PDF) and Bacillus thermoproteolyticus thermolysin (TLN) are two representative metal-requiring peptidases having remarkably similar active centers but distinctively different metal preferences. Zinc is a competent catalytic cofactor for TLN but not for PDF. Reaction pathways and the associated energetics for both enzymes were determined using combined semiempirical and ab initio quantum mechanical/molecular mechanical modeling, without presuming reaction coordinates. The results confirmed that both enzymes catalyze via the same chemical steps, and reproduced their different preferences for zinc or iron as competent cofactors. Further analyses indicated that different feasibility of the nucleophilic attack step leads to different metal preferences of the two enzymes. In TLN, the substrate is strongly activated and can serve as the fifth coordination ligand of zinc prior to the chemical steps. In PDF, the substrate carbonyl is activated by the chemical step itself, and becomes the fifth coordination partner of zinc only in a later stage of the nucleophilic attack. These leads to a much more difficult nucleophilic attack in PDF than in TLN. Different from some earlier suggestions, zinc has no difficulty in accepting an activated substrate as the fifth ligand to switch from tetra- to penta-coordination in either PDF or TLN. When iron replaces zinc, its stronger interaction with the hydroxide ligand may lead to higher activation barrier in TLN. In PDF, the stronger interactions of iron with ligands allow iron-substrate coordination to take place either before or at a very early stage of the chemical step, leading to effective catalysis. Our calculations also show combined semiempirical and ab initio quantum mechanical modeling can be efficient approaches to explore complicated reaction pathways in enzyme systems.     

Structures and hydration enthalpies of cationized glutamine and structural analogues in the gas phase

The structures of lithiated and sodiated glutamine, both with and without a water molecule, are investigated using experiment and theory. Loss of water from these complexes and from lithiated and sodiated complexes of asparagine methyl ester, asparagine ethyl ester, and glutamine methyl ester is probed with blackbody infrared radiative dissociation experiments performed over a wide temperature range. Threshold dissociation energies, E(o), for loss of a water molecule from these complexes are obtained from master equation modeling of these data. The values of E(o) are 63 +/- 1 and 53 +/- 1 kJ/mol for the lithiated and sodiated glutamine complexes, respectively. These values are similar to those for the nonzwitterionic model complexes and are in excellent agreement with calculated values. In contrast, water binding to the zwitterionic form is calculated to be significantly higher. These results indicate that glutamine in these lithiated and sodiated complexes with a water molecule are nonzwitterionic. Complexes with the asparagine side chain have slightly higher E(o) values than those with the glutamine side chain, a result consistent with more effective solvation of the metal ion due to the slightly longer side chain of glutamine. Calculations indicate that lithiated and sodiated glutamine are nonzwitterionic, with the metal ion interacting with the amine nitrogen and carbonyl oxygen from the amino acid backbone and the amide oxygen of the side chain. Addition of a water molecule does not affect the lowest-energy structure of lithiated glutamine, whereas, for sodiated glutamine, the lowest-energy zwitterionic and nonzwitterionic structures are essentially isoenergetic.     

Solvothermal Synthesis and Crystal Structure of One‐Dimensional Chains of Anhydrous Zinc and Magnesium Formate

Reaction of the zinc or magnesium formate dihydrates in formic acid under solvothermal conditions results in the formation of single crystals of the anhydrous metal(II) formates β‐Zn(OOCH)₂ and β‐Mg(OOCH)₂. Both structures form one‐dimensional chains of μ‐oxygen‐bridged metal atoms. Single crystal diffraction studies reveal that β‐zinc formate represents the first structure in which chains of oxygen‐bridged metal atoms are connected by alternating single, double and triple oxygen atom bridges resulting in the first observation of corner, edge and face sharing coordination octahedra within a single chain. Polycrystalline material can be obtained by dehydration reaction of zinc formate dihydrate. β‐magnesium formate is the crystalline product that is obtained by annealing the amorphous intermediate phase after dehydration of magnesium formate dihydrate.