Catalina Achim

Evidence for a Near-Resonant Charge Transfer Mechanism for Double-Stranded Peptide Nucleic Acid

We present evidence for a near-resonant mechanism of charge transfer in short peptide nucleic acid (PNA) duplexes obtained through electrochemical, STM break junction (STM-BJ), and computational studies. A seven base pair (7-bp) PNA duplex with the sequence (TA)(3)-(XY)-(TA)(3) was studied, in which XY is a complementary nucleobase pair. The experiments showed that the heterogeneous charge transfer rate constant (k(0)) and the single-molecule conductance (σ) correlate with the oxidation potential of the purine base in the XY base pair. The electrochemical measurements showed that the enhancement of k(0) is independent, within experimental error, of which of the two PNA strands contains the purine base of the XY base pair. 7-bp PNA duplexes with one or two GC base pairs had similar measured k(0) and conductance values. While a simple superexchange model, previously used to rationalize charge transfer in single stranded PNA (Paul et al. J. Am. Chem. Soc. 2009, 131, 6498-6507), describes some of the experimental observations, the model does not explain the absence of an enhancement in the experimental k(0) and σ upon increasing the G content in the duplexes from one to two. Moreover, the superexchange model is not consistent with other studies (Paul et al. J. Phys. Chem. B 2010, 114, 14140), that showed a hopping charge transport mechanism is likely important for PNA duplexes longer than seven base pairs. A quantitative computational analysis shows that a near-resonant charge transfer regime, wherein a mix of superexchange and hopping mechanisms are expected to coexist, can rationalize all of the experimental results.

Role of Nucleobase Energetics and Nucleobase Interactions in Single-Stranded Peptide Nucleic Acid Charge Transfer

Self-assembled monolayers of single-stranded (ss) peptide nucleic acids (PNAs) containing seven nucleotides (TTTXTTT), a C-terminus cysteine, and an N-terminus ferrocene redox group were formed on gold electrodes. The PNA monomer group (X) was selected to be either cytosine (C), thymine (T), adenine (A), guanine (G), or a methyl group (Bk). The charge transfer rate through the oligonucleotides was found to correlate with the oxidation potential of X. Kinetic measurements and computational studies of the ss-PNA fragments show that a nucleobase mediated charge transport mechanism in the deep tunneling superexchange regime can explain the observed dependence of the kinetics of charge transfer on the PNA sequence. Theoretical analysis suggests that the charge transport is dominantly hole-mediated and takes place through the filled bridge orbitals. The strongest contribution to conductance comes from the highest filled orbitals (HOMO, HOMO-1, and HOMO-2) of individual bases, with a rapid drop off in contributions from lower lying filled orbitals. Our studies further suggest that the linear correlation observed between the experimental charge transfer rates and the oxidation potential of base X arises from weak average interbase couplings and similar stacking geometries for the four TTTXTTT systems.

The Single-Molecule Conductance and Electrochemical Electron-Transfer Rate Are Related by a Power Law

This study examines quantitative correlations between molecular conductances and standard electrochemical rate constants for alkanes and peptide nucleic acid (PNA) oligomers as a function of the length, structure, and charge transport mechanism. The experimental data show a power-law relationship between conductances and charge transfer rates within a given class of molecules with the same bridge chemistry, and a lack of correlation when a more diverse group of molecules is compared, in contrast with some theoretical predictions. Surprisingly, the PNA duplexes exhibit the lowest charge-transfer rates and the highest molecular conductances. The nonlinear rate-conductance relationships for structures with the same bridging chemistries are attributed to differences in the charge-mediation characteristics of the molecular bridge, energy barrier shifts and electronic dephasing, in the two different experimental settings.

Solution structure of a peptide nucleic acid duplex from NMR data: features and limitations.

This paper describes the results of a 1D and 2D NMR spectroscopy study of a palindromic 8-base pair PNA duplex GGCATGCC in H2O and H2O-D2O solutions. The (1)H NMR peaks have been assigned for most of the protons of the six central base pairs, as well as for several amide protons of the backbone. The resulting 36 interbase and base-backbone distance restraints were used together with Watson-Crick restraints to generate the PNA duplex structure in the course of 10 independent simulated annealing runs followed by restrained molecular dynamics (MD) simulations in explicit water. The resulting PNA structures correspond to a P-type helix with helical parameters close to those observed in the crystal structures of PNA. Based on the current limited number of restraints obtained from NMR spectra, alternative structures obtained by MD from starting PNA models based on DNA cannot be ruled out and are also discussed.

Effect of Backbone Flexibility on Charge Transfer Rates in Peptide Nucleic Acid Duplexes

Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.

Metal Binding to Ligand-Containing Peptide Nucleic Acids

The substitution of nucleobases in nucleic acid duplexes with ligands that have high affinity for transition metal ions creates metal-binding sites at specific locations within the duplexes. Several studies on the incorporation of metal ions into DNA and peptide nucleic acid (PNA) duplexes have suggested that the stability constant of the metal complex formed within the duplexes is a primary determinant of the thermal stability of the duplexes. To understand this relationship, we have synthesized two PNA monomers that carry the same ligand, namely 8-hydroxyquinoline, but have this ligand attached differently to the PNA backbone. The PNA monomers have been incorporated into PNA duplexes. UV and CD spectroscopy and calorimetric studies of the 8-hydroxyquinoline-PNA duplexes showed that the effect of the stability of the metal complex on the PNA duplexes was significantly modulated by the steric relationship between the complex and the duplex. This information is useful for the construction of hybrid inorganic-nucleic acid nanostructures.

EXAFS and Mössbauer characterization of the Diiron(III) site in stearoyl-acyl carrier protein Δ9– desaturase

Abstract Stearoyl-acyl carrier protein (ACP) Δ9-desaturase (Δ9D) from the castor plant is the best characterized soluble acyl-ACP desaturase. This enzyme utilizes a diiron center to catalyze the O2- and NADPH-dependent introduction of a cis double bond between carbons 9 and 10 of stearoyl-ACP, yielding oleoyl-ACP. In the present study, we have used X-ray absorption spectroscopy to provide the first metrical information for the diferric oxidation state. These studies reveal distinct diiron clusters that have Fe-Fe distances of either 3.12 or 3.41 Å. The species having the 3.12 Å Fe-Fe distance also exhibits a 1.8 Å Fe-O bond and is thus proposed to represent Δ9D molecules containing a (μ-oxo)bis(μ-carboxylato)diiron(III) cluster. The species having the 3.41 Å Fe-Fe distance exhibits no short Fe-O bond, and thus likely represents Δ9D molecules containing a (μ-hydroxo)diiron(III) cluster. Mössbauer studies of the extended X-ray absorption fine structure (EXAFS) samples revealed three quadrupole doublets (ΔEQ(1)=1.53 mm/s, 72%;ΔEQ(2)=0.72 mm/s, 21%;ΔEQ(3)=2.20 mm/s, 7%) that originate from three distinct dinuclear clusters. From analysis of spectral intensities and by comparison with previous studies of (μ-oxo)- and (μ-hydroxo)diiron(III) clusters in both model complexes and proteins, doublet 1, the Mössbauer majority species, is likely associated with the EXAFS majority species having a 3.12 Å Fe-Fe separation and a 1.8 Å Fe-μ-oxo bond, while doublet 2 likely results from one iron site (or both) of a cluster associated with the EXAFS species having a 3.41 Å Fe-Fe separation. The presence of multiple diiron center conformations in diferric Δ9D may reflect the necessity for the active site to allow access of the substrate stearoyl-ACP (∼9 kDa) during desaturation catalysis.

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD studies of the wild-type protein

Abstract Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D=1.2 cm−1, that are about 40% smaller than the D-value of the wild-type protein. The 57Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has δ=0.24±0.01 mm/s at 4.2 K rather than δ=0.32 mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts δ=0.79 mm/s at 4.2 K, consistent with tetrahedral Fe2+(Cys)3(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D=−7±1 cm−1, E/D=0.09 for form A and D=+6.2±1.3 cm−1, E/D=0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained gz=2.08±0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77 K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe2+ site. Studies in different buffers in the pH 6–9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30–50% v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700 cm−1. Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300 cm−1; a third d-d transition, if present, must occur with energy lower than 3300 cm−1. The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100 cm−1 in form A and 5350, 6380 cm−1 in form B.

The crystal structure of non-modified and bipyridine-modified PNA duplexes.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)(2), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 Å, respectively. The non-modified PNA duplex adopts a P-type helical structure similar to that of previously characterized PNAs. The atomic-level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P-type helical structure, its flexibility is relatively high. For example, the base-pair rise in the bipyridine-containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π-stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl-modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.

Investigation of Phthalocyanine Catalysts for the Aerobic Synthesis of meso-Substituted Porphyrins

The aerobic oxidation process for the synthesis of porphyrins, previously performed using 5 mol % p-chloranil (TCQ), 5 mol % iron(II) phthalocyanine (FePc) and stoichiometric amounts of O2, has been refined using new phthalocyanine catalysts. Four phthalocyanine catalysts have been prepared, characterized by Mossbauer spectroscopy and examined for efficacy in the high concentration (0.1 M) synthesis of tetraphenylporphyrin at room temperature. Each phthalocyanine has been identified to be a μ-oxo dimer. Two catalysts are soluble (the μ-oxo dimers [(t-butyl)4FePc]2O and [(n-C6H13O)4FePc]2O) and enable homogeneous reactions, while two are insoluble (the μ-oxo(1) and μ-oxo(2) dimers of FePc, (FePc)2O) and give heterogeneous reactions. These four phthalocyanine compounds provide efficient catalysis at the 0.3–1 mol % level using only 1 mol % quinone or hydroquinone (TCQ, DDQ, TCQH2 or DDQH2), affording ~25% yields of tetraphenylporphyrin in 60 min of oxidation. There are no discernible advantages of the homogeneous versus heterogeneous catalysts. The μ-oxo dimers are active, but FePc is inactive, at the 0.3 mol % level. The activity of the FePc sample at the 5 mol % level is attributed to residual μ-oxo dimer impurity. This aerobic oxidation process is superior to stoichiometric oxidation with TCQ or DDQ, and can be performed in the presence of BF3·O(Et)2, trifluoroacetic acid, or under neutral conditions.