Intramolecular Electron Transfer Rate Constants Research Articles

Modulation of the rate of reversible electron transfer in oxidized tryptophan and tyrosine containing peptides in acidic aqueous solution.

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) was used to investigate reversible intramolecular electron transfer (IET) in short-lived oxidized peptides, which had different structures and contained tryptophan and tyrosine residues, in an acidic aqueous solution with a pH below the pKa of the tryptophanyl cation radical. The CIDNP kinetic data were obtained at the microsecond scale and were analyzed in detail to calculate the rate constants for electron transfer in both directions: from the tyrosine residue to the tryptophanyl cation radical, kf, and from the tryptophan residue to the neutral tyrosyl radical, kr. The charge of the terminal amino group and the presence of glycine and proline spacers were shown to strongly affect the rate constants of the reaction under study. Among these functional groups, the presence and the location of the positive charge on the amino group in close proximity to the cationic indolyl radical had the strongest effect on the rate constant of the forward IET from the tyrosine residue to the tryptophanyl radical cation, kf. This effect was manifested as an increase of 2 orders of magnitude in kf for a change in the linkage order between residues in the dipeptide: kf = 4 × 10(3) s(-1) for the oxidized Tyr-Trp increased to kf = 5.5 × 10(5) s(-1) in oxidized Trp-Tyr. The reverse rate constant for IET was less sensitive to the amino group charge. Moreover, the presence of glycine or proline spacers in the peptides with a tryptophan residue at the N-terminus not only reduced the IET rate constant but also shifted the equilibrium of the IET in the reaction under study toward the formation of tyrosyl radicals with respect to the peptide Trp-Tyr. That is, the glycine or proline spacers affected the difference in the reduction potential of the tryptophanyl and tyrosyl radicals.

Calculation from First Principles of Intramolecular Golden-Rule Rate Constants for Photo-Induced Electron Transfer in Molecular Donor–Acceptor Systems

The feasibility of calculating photoinduced intramolecular electron transfer rate constants in realistic molecular donor–acceptor systems via Fermi’s golden rule, using inputs obtained from state-of-the-art electronic structure techniques, is demonstrated and tested. To this end, calculations of photoinduced electron transfer rate constants were performed on two benchmark systems: (1) phenylacetylene-bridged carbazole-naphthalimide (meta and para) and (2) C60-(N,N-dimethylaniline). Intramolecular input parameters such as normal-mode frequencies, Huang–Rhys factors, and electronic coupling coefficients were obtained via ground state, time-dependent, and constrained density functional theory. Good agreement between the intramolecular Fermi’s golden rule rate constants and the experimental rate constants is found for both systems without accounting for the solvent reorganization. The relative roles of intramolecular vs intermolecular modes at promoting electron transfer and the validity of several limits of Fermi’s golden rule for describing intramolecular electron transfer are discussed.

Probing the role of a conserved salt bridge in the intramolecular electron transfer kinetics of human sulfite oxidase

Sulfite oxidase (SO) is a vital metabolic enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed mechanism of this molybdenum cofactor dependent enzyme involves two one-electron intramolecular electron transfer (IET) steps from the molybdenum center to the iron of the b 5-type heme and two one-electron intermolecular electron transfer steps from the heme to cytochrome c. This work focuses on how the electrostatic interaction between two conserved amino acid residues, R472 and D342, in human SO (hSO) affects catalysis. The hSO variants R472M, R472Q, R472K, R472D, and D342K were created to probe the effect of the position of the salt bridge charges, along with the interaction between these two residues. With the exception of R472K, these variants all showed a significant decrease in their IET rate constants, k et, relative to wild-type hSO, indicating that the salt bridge between residues 472 and 342 is important for rapid IET. Surprisingly, however, except for R472K and R472D, all of the variants show k cat values higher than their corresponding k et values. The turnover number for R472D is about the same as k et, which suggests that the change in this variant is rate-limiting in catalysis. Direct spectroelectrochemical determination of the Fe(III/II) reduction potentials of the heme and calculation of the Mo(VI/V) potentials revealed that all of the variants affected the redox potentials of both metal centers, probably due to changes in their environments. Thus, the position of the positive charge of R472 and that of the negative charge of D342 are both important in hSO, and changing either the position or the nature of these charges perturbs IET and catalysis.

Designed azurins show lower reorganization free energies for intraprotein electron transfer

Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.

Intramolecular electron transfer in sulfite-oxidizing enzymes: probing the role of aromatic amino acids

Sulfite oxidase (SO) is a molybdoheme enzyme that is important in sulfur catabolism, and mutations in the active site region are known to cause SO deficiency disorder in humans. This investigation probes the effects that mutating aromatic residues (Y273, W338, and H337) in the molybdenum-containing domain of human SO have on both the intramolecular electron transfer (IET) rate between the molybdenum and iron centers using laser flash photolysis and on catalytic turnover via steady-state kinetic analysis. The W338 and H337 mutants show large decreases in their IET rate constants (k (ET)) relative to the wild-type values, suggesting the importance of these residues for rapid IET. In contrast, these mutants are catalytically competent and exhibit higher k (cat) values than their corresponding k (ET), implying that these two processes involve different conformational states of the protein. Redox potential investigations using spectroelectrochemistry revealed that these aromatic residues close to the molybdenum center affect the potential of the presumably distant heme center in the resting state (as shown by the crystal structure of chicken SO), suggesting that the heme may be interacting with these residues during IET and/or catalytic turnover. These combined results suggest that in solution human SO may adopt different conformations for IET and for catalysis in the presence of the substrate. For IET the H337/W338 surface residues may serve as an alternative-docking site for the heme domain. The similarities between the mutant and wild-type EPR spectra indicate that the active site geometry around the Mo(V) center is not changed by the mutations studied here.

Tunneling versus Hopping in Mixed-Valence Oligo-p-phenylenevinylene Polychlorinated Bis(triphenylmethyl) Radical Anions

Radical anions 1(-•)-5(-•), showing different lengths and incorporating up to five p-phenylenevinylene (PPV) bridges between two polychlorinated triphenylmethyl units, have been prepared by chemical or electrochemical reductions from the corresponding diradicals 1-5 which were prepared using Wittig-Horner-type chemistry. Such radical anions enabled us to study, by means of UV-vis-NIR and variable-temperature electron spin resonance spectroscopies, the long-range intramolecular electron transfer (IET) phenomena in their ground states, probing the influence of increasing the lengths of the bridges without the need of using an external bias to promote IET. The temperature dependence of the IET rate constants of mixed-valence species 1(-•)-5(-•) revealed the presence of two different regimes at low and high temperatures in which the mechanisms of electron tunneling via superexchange and thermally activated hopping are competing. Both mechanisms occur to different extents, depending on the sizes of the radical anions, since the lengths of the oligo-PPV bridges notably influence the tunneling efficiency and the activation energy barriers of the hopping processes, the barriers diminishing when the lengths are increased. The nature of solvents also modifies the IET rates by means of the interactions between the oligo-PPV bridges and the solvents. Finally, in the shortest compounds 1(-•) and 2(-•), the IET induced optically through the superexchange mechanism can also be observed by the exhibited intervalence bands, whose intensities decrease with the length of the PPV bridge.

Intramolecular Electron Transfer in Sulfite-Oxidizing Enzymes: Elucidating the Role of a Conserved Active Site Arginine

All reported sulfite-oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. To improve our understanding of the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for improving our understanding of residue function in IET without the added complexity of the interdomain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) exhibited increased IET rate constants relative to that of the wild type (3-4-fold) when studied by laser flash photolysis. The gain in function observed in SDH(R55Q) and SDH(R55M) suggests that the reduction in the level of IET seen in HSO(R160Q) is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the interdomain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDH(WT), SDH(R55M), and SDH(R55Q) show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (K(eq)) and rate constants (k(et)) of both variants with respect to the SDH(WT) enzyme. SDH(WT) has a k(et) that is independent of pH and a K(eq) that increases as pH decreases; on the other hand, both SDH(R55M) and SDH(R55Q) have a k(et) that increases as pH decreases, and SDH(R55M) has a K(eq) that is pH-independent. IET in the SDH(R55Q) variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from that of SDH(WT), but which also occurs in HSO. IET in SDH(R55K) is slower than in SDH(WT). A new analysis of the possible mechanistic pathways for sulfite-oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.

Kinetic studies on the reaction of N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetate chromium(III) with hydrogen peroxide

The rate of reaction of Cr(III)L (L=N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetate) with hydrogen peroxide was studied in aqueous media and was found to yield Cr(VI) over the temperature range of 25–41 °C. The reaction was followed spectrophotometrically at 396 nm under pseudo-first order conditions with hydrogen peroxide in a large excess. The reaction follows first-order kinetics in Cr(III). The dependence of the rate constant on hydrogen peroxide concentration is attributed to the formation of an intermediate between the monohydroxy chromium(III) complex and hydrogen peroxide which decomposes in the rate determining step. At high hydrogen peroxide concentration, the order is changed from first to zero order.The values of the intramolecular electron transfer rate constant, the formation constant of the intermediate complex and the activation parameters were calculated.

The first example of photochemical reduction of nitrite into nitrogen monoxide by a dinuclear Ru(ii)–Cu(ii) complex and photoinduced intramolecular electron transfer reaction between Ru(ii) and Cu(ii) moieties

The photochemical reduction of nitrite to NO by the dinuclear Ru(II)-Cu(II) complex ([Ru(bpy)(2)(Mebpy-COOC(3)H(6))Me(2)bpaCu(H(2)O)(ClO(4))](ClO(4)).(PF(6))(2).(H(2)O) was observed in the absence of sacrificial electron donor reagents in CH(2)Cl(2). The reaction rate of the photoreduction of nitrite depended upon the concentration of the excited Ru(II) moiety in the complex. The photoinduced intramolecular electron transfer rate constants between the Ru(II) and Cu(II) moieties (*Ru(II)-Cu(II) --> Ru(III)-Cu(I) and Ru(III)-Cu(I) --> Ru(II)-Cu(II)) in the dinuclear complex were calculated to be 2.3 x 10(9) and 8.3 x 10(7) s(-1), respectively, by laser flash photolysis.

Kinetics and mechanism of oxidation of the binary and ternary complexes of chromium(III) involving inosine and glycine by N -bromosuccinimide

The kinetics of oxidation of the chromium(III) complexes, [Cr(Ino)(H2O)5]3+ and [Cr(Ino)(Gly)(H2O)3]2+ (Ino = Inosine and Gly = Glycine) involving a ligands of biological significance by N-bromosuccinimide (NBS) in aqueous solution to chromium(VI) have been studied spectrophotometrically over the 25–45°C range. The reaction is first order with respect to both [NBS] and [Cr], and increases with pH over the 6.64–7.73 range in both cases. The experimental rate law is consistent with a mechanism in which the hydroxy complexes [Cr(Ino)(H2O)4(OH)]2+ and [Cr(Ino)(Gly)(H2O)2(OH)]+ are significantly more reactive than their conjugate acids. The value of the intramolecular electron transfer rate constant, k 1, for the oxidation of the [Cr(Ino)(H2O)5]3+ (6.90 × 10−4 s−1) is lower than the value of k 2 (9.66 × 10−2 s−1) for the oxidation of [Cr(Ino)(Gly)(H2O)2]2+ at 35°C and I = 0.2 mol dm−3. The activation parameters have been calculated. Electron transfer apparently takes place via an inner-sphere mechanism. †ed from the MSc thesis of Mohamed A. Nagdy.

Solvent Dynamical Control of Ultrafast Ground State Electron Transfer: Implications for Class II−III Mixed Valency

We relate the solvent and temperature dependence of the rates of intramolecular electron transfer (ET) of mixed valence complexes of the type {[Ru3O(OAc)6(CO)(L)]2-BL}-1, where L = pyridyl ligand and BL = pyrazine. Complexes were reduced chemically or electrochemically to obtain the mixed valence anions in seven solvents: acetonitrile, methylene chloride, dimethylformamide, tetrahydrofuran, dimethylsulfoxide, chloroform, and hexamethylphosphoramide. Rate constants for intramolecular ET were estimated by simulating the observed degree of nu(CO) IR band shape coalescence in the mixed valence state. Correlations between rate constants for ET and solvent properties including static dielectric constant, optical dielectric constant, the quantity 1/epsilonop - 1/epsilonS, microscopic solvent polarity, viscosity, cardinal rotational moments of inertia, and solvent relaxation times were examined. In the temperature study, the complexes displayed a sharp increase in the ket as the freezing points of the solvents methylene chloride and acetonitrile were approached. The solvent phase transition causes a localized-to-delocalized transition in the mixed valence ions and an acceleration in the rate of ET. This is explained in terms of decoupling the slower solvent motions involved in the frequency factor nuN which increases the value of nuN. The observed solvent and temperature dependence of the ket for these complexes is used in order to formulate a new definition for Robin-Day class II-III mixed valence compounds. Specifically, it is proposed that class II-III compounds are those for which thermodynamic properties of the solvent exert no control over ket, but the dynamic properties of the solvent still influence ket.

Electron transfer at the class II/III borderline of mixed valency: dependence of rates on solvent dynamics and observation of a localized-to-delocalized transition in freezing solvents

The dependence of the rates of intramolecular electron transfer (ET) of mixed-valence complexes of the type {[Ru3O(OAc)6(CO)(L)]2-BL}-1, where L is the pyridyl ligand and BL is the pyrazine on solvent type and temperature is described. Complexes were reduced chemically to obtain the mixed-valence anions in acetonitrile (CH3CN) and methylene chloride (CH2Cl2). Rate constants for intramolecular ET were estimated by simulating the observed degree of nu(CO) infrared (IR) bandshape coalescence in the mixed-valence state. In the strongly coupled mixed-valence states of these complexes, the electronic coupling, HAB, approaches lambda/2, where lambda is the total reorganization energy. The activation energy is thus nearly zero, and rate constants are in the 'ultrafast' regime where they depend on the pre-exponential terms within the frequency factor, nuN. The frequency factor contains both external (solvent dynamics) and internal (molecular vibrations) contributions. In general, external solvent motions are slower than internal vibrations, and therefore control ET rates in fluid solution. A profound increase in the degree of nu(CO) IR bandshape coalescence is observed as the temperature approaches the freezing points of the solvents methylene chloride (f.p. -92 degrees C) and acetonitrile (f.p. -44 degrees C). Decoupling the slower solvent motions involved in the frequency factor nuN for ET by freezing the solvent causes a transition from solvent dynamics to internal vibration-limited rates. The solvent phase transition causes a localized-to-delocalized transition in the mixed-valence ions that accelerates the rate of ET.

Relationships between Nonadiabatic Bridged Intramolecular, Electrochemical, and Electrical Electron-Transfer Processes

Fermi's golden rule is used to develop relationships between rate constants for electron transfer in donor-bridge-acceptor and electrode-bridge-acceptor systems and resistances across metal-bridge-electrode and metal-bridge-tip junctions. Experimental data on electron-transfer rates through alkanethiolate, oligophenylene, and DNA bridges are used to calculate the electronic coupling matrix element per state through these moieties. The formulation is then used to predict the resistance of these bridges between two gold contacts. This approach provides a straightforward method for experimentalists to assess the self-consistency between intramolecular electron-transfer rate constants and low-bias resistances measured for molecularly bridged junctions between two metallic contacts. Reported resistances for alkanethiolate bridges vary by a factor of 20, with predicted resistances falling within this range. However, comparisons between carboxylato and directly linked alkanethiolate bridges suggest differences between the coupling at the interface to either the redox center or the gold electrode in such systems. Calculated resistances for oligophenylene bridges are close to those measured experimentally in a similar oligophenylene system.

Comparisons of Measured Rate Constants with Spectroscopically Determined Electron-Transfer Parameters

This work involves comparison of rate constants measured for an intervalence (IV) compound with electron-transfer parameters derived from its optical absorption spectrum. The temperature-dependent rate constants for the radical cation having 3-tert-butyl-2,3-diazabicyclo[2.2.2]oct-2-yl (hydrazine) charge-bearing units attached para to a tetramethylbenzene bridge (1+) were previously measured. In this study, resonance Raman is used to calculate the magnitudes of the distortions of normal modes of vibration caused by excitation into the intervalence absorption band. These data produce a vibrational reorganization energy lambdavsym of 9250 cm(-1), and averaged single-mode omegav for use in the Golden Rule equation of 697 cm(-1). Zhu-Nakamura theory has been used to calculate preexponential factors for analysis of the previously measured variable temperature optical spectra using quartic-enhanced intervalence bands to extract the total reorganization energy and the intramolecular electron-transfer rate constants for intramolecular electron transfer using electron spin resonance. In contrast to using the Golden Rule equation, separation of lambda into solvent and vibrational components is not significant for these data. The Zhu-Nakamura theory calculations produce ln(k/T) versus 1/T slopes that are consistent with the experimental data for electronic couplings that are somewhat larger than the values obtained from the optical spectra using Hush's method.

Novel Bifunctional Viologen-Linked Pyrene Conjugates: Synthesis and Study of Their Interactions with Nucleosides and DNA

With the objective of developing efficient DNA oxidizing agents, a new series of viologen-linked pyrene conjugates with the general formula PYLnV(2+), having a different number of methylene spacer units (Ln) was synthesized, and their interactions with nucleosides and DNA have been investigated through photophysical and biophysical techniques. The viologen-linked pyrene derivatives PYL1V(2+) (n =equals; 1), PYL7V(2+) (n = 7), and PYL12V(2+) (n = 12) exhibited characteristic fluorescence emission of the pyrene chromophore centered around 380 nm but with significantly reduced yields when compared to those of the model compound PYL1Et(3)(+). The fluorescence quenching observed in these systems is explained through an electron-transfer mechanism based on a calculated favorable change in free energy (DeltaG(ET) = -1.59 eV), and the redox species characterized through laser flash photolysis studies. Intramolecular electron-transfer rate constants (k(ET)) were calculated from the observed fluorescence yields, and the singlet lifetimes of the model compound and are found to decrease with increasing spacer length. The DNA binding studies of these systems through photophysical, chiroptical, and viscometric techniques demonstrated that these systems effectively undergo DNA intercalation with association constants (KDNA) in the range of 1.1-2.6 x 10(4) M(-1) and exhibit 2:1 sequence selectivity for poly(dG) x poly(dC) over poly(dA) x poly(dT). Photoactivation of these systems initiates electron transfer from the singlet excited state of the pyrene chromophore to the viologen moiety followed by an electron transfer from DNA to the oxidized pyrene. This results in the formation of stable charge-separated species such as radical cations of both DNA and reduced viologen as characterized by laser flash photolysis studies and subsequently the oxidized DNA modifications. These novel systems are soluble in buffer media, stable under irradiation conditions, and oxidize DNA efficiently and selectively through a cosensitization mechanism and hence can be useful as photoactivated DNA cleaving agents.

Intramolecular Electron Transfer in Lysozyme Studied by Time-Resolved Chemically Induced Dynamic Nuclear Polarization

The kinetics of the chemically induced dynamic nuclear polarization (CIDNP) produced in reactions of hen lysozyme with photosensitizers have been studied for the native state of the protein at pH 3.8 and for two denatured states. The latter were generated by raising the temperature to 80 degrees C or by combining a temperature rise (to 50 degrees C) with the addition of chemical denaturant (10 M urea). Detailed analysis of the CIDNP time dependence on a microsecond time scale revealed that, in both denatured states, intramolecular electron transfer (IET) from a tyrosine residue to the cation radical of a tryptophan residue (rate constant k(f)) is highly efficient and plays a decisive role in the evolution of the nuclear polarization. To describe the observed CIDNP kinetics with a self-consistent set of parameters, IET in the reverse direction, from a tryptophan residue to a tyrosine residue radical (rate constant k(r)), has also to be taken into account. The IET rate constants determined by analysis of the CIDNP kinetics are, at 80 degrees C: k(f) = 1 x 10(5) s(-1) and k(r) = 1 x 10(4) s(-1); at 50 degrees C in the presence of 10 M urea: k(f) = 7 x 10(4) s(-1), k(r) = 1 x 10(4) s(-1). IET does not appear to influence the CIDNP kinetics of the native state.

The Pathogenic Human Sulfite Oxidase Mutants G473D and A208D Are Defective in Intramolecular Electron Transfer

Mutations G473D and A208D were identified in patients with isolated sulfite oxidase (SO) deficiency, and the equivalent amino acids (G451 and A186, respectively) have been localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garrett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983]. To assess the effects of these mutations in human SO, steady-state kinetic studies of enzyme turnover and laser flash photolysis measurements of intramolecular electron transfer (IET) rate constants between the reduced heme [Fe(II)] and Mo(VI) centers were carried out in the recombinant G473D, G473A, G473W, G473D/R212A, and A208D human SO mutants. In the G473D and A208D mutants, the IET rate constants at pH 6.0 are decreased by 3 orders of magnitude relative to that of the wild type. Steady-state kinetic measurements indicate that the IET process is the rate-limiting step in the catalytic cycle of these two mutants. Thus, the large decreases in the IET rate constants and the kcat values, and the large increases in the Km(sulfite) values, rationalize the fatal impact of these mutations. Far-UV CD spectra of G473D indicate that the protein backbone conformation is remarkably changed, and the sedimentation equilibrium indicates that the protein is monomeric. Furthermore, EPR studies also suggest that the active site structure of the Mo(V) form of A208D is different from that of the wild type. In contrast, similar studies on G473A show that it is dimeric, that its Mo(V) active site structure is similar to that of the wild type, and that its IET rate constant is only 2.6-fold smaller than that of the wild type. IET in G473W is severely impaired, and no IET is observed for G473D/R212A. In chicken SO, the equivalent residues (G451 and A186) are both buried inside the protein. Thus, for human SO, the mutations to charged residues at the equivalent sites most likely cause crucial global or localized structural changes, and expose an alternative docking site that may compete with the Mo domain for docking of the heme, thereby retarding IET and efficient catalytic turnover of the sulfite oxidation reaction.

Electrochemical activation of glucose oxidase with a 140-fold enhancement in intramolecular electron transfer rate constant

In this work, we describe the electrochemical activation of glucose oxidase (GOx) via covalent attachment of a novel redox mediator, Os(bpy)2(API)Cl (bpy = 2,2'-bipyridine, API = 3-aminopropylimidazole), to the peptide backbone of GOx targeting at aspartate and glutamate residues. Cyclic voltammetry showed a pair of well-defined voltammetric peaks centered at 0.11 V for the activated enzyme. Os(bpy)2(API)Cl promotes direct oxidation of FADH2 centers in GOx without the need of any mediating agents in solution. Amperometric tests in glucose solution revealed that the GOx retains its enzymatic activity toward the oxidation of glucose. An intramolecular electron transfer rate constant of 1.0x10(5) s(-1) was obtained for the activated GOx, compared with the rate constant of 7.0x10(2) s(-1) of the natural GOx-oxygen system, making this an amenable system for biosensor applications. Attempts were made in utilizing the activated GOx as an electrochemical tag in nucleic acid assay.

Design of photoactivated DNA oxidizing agents: synthesis and study of photophysical properties and DNA interactions of novel viologen-linked acridines.

A new series of photoactivated DNA oxidizing agents in which an acridine moiety is covalently linked to viologen by an alkylidene spacer was synthesized, and their photophysical properties and interactions with DNA, including DNA cleaving properties, were investigated. The fluorescence quantum yields of the viologen-linked acridines were found to be lower than that of the model compound 9-methylacridine (MA). The changes in free energy for the electron transfer reactions were found to be favorable, and the fluorescence quenching observed in these systems is explained by an electron transfer mechanism. Intramolecular electron transfer rate constants were calculated from the observed fluorescence quantum yields and singlet lifetime of MA and are in the range from 1.06x10(10) s(-1) for 1 a (n=1) to 6x10(8) s(-1) for 1 c (n=11), that is, the rate decreases with increasing spacer length. Nanosecond laser flash photolysis of these systems in aqueous solutions showed no transient absorption, but in the presence of guanosine or calf thymus DNA, transient absorption due to the reduced viologen radical cation was observed. Studies on DNA binding demonstrated that the viologen-linked acridines bind effectively to DNA in both intercalative and electrostatic modes. Results of PM2 DNA cleavage studies indicate that, on photoexcitation, these molecules induce DNA damage that is sensitive to formamidopyrimidine DNA glycosylase. These viologen-linked acridines are quite stable in aqueous solutions and oxidize DNA efficiently and hence can be useful as photoactivated DNA-cleaving agents which function purely by the co-sensitization mechanism.

Role of Conserved Tyrosine 343 in Intramolecular Electron Transfer in Human Sulfite Oxidase

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.