The journal of physical chemistry. B, Jan 18, 2014
Protein conformational heterogeneity and dynamics are known to play an important role in enzyme c... more Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm(-1), respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases a...
Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicot... more Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD(+)·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C2═O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2═O group and active site residues, as shown by the observation of at least four C2═O stretch bands indicating varying degrees of C2═O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.
Aryl diphosphenes (Ar-P=P-Ar) possess features that may make them useful in photonic devices, inc... more Aryl diphosphenes (Ar-P=P-Ar) possess features that may make them useful in photonic devices, including the possibility for photochemical E-Z isomerization. Development of good models guided by computations is hampered by poor correspondence between predicted and experimental UV/vis absorption spectra. An hypothesis that the phenyl twist angle (i.e. PPCC torsion) accounts for this discrepancy is explored, with positive findings. DFT and TDDFT (B3LYP) were applied to the phenyl-P=P-phenyl (Ph-P=P-Ph) model compound over a range of phenyl twist angles, and to the Ph-P=P-Ph cores of two crystallographically characterized diphosphenes: bis-(2,4,6-tBu 3 C 6 H 2 )diphosphene (Mes*-P=P-Mes*) and bis-(2,6-Mes 2 C 6 H 3 )-diphosphene (Dmp-P=P-Dmp). A shallow PES is observed: the full range of phenyl twist angles is accessible for under 5 kcal/mol. The Kohn-Sham orbitals (KS-MOs) exhibit stabilization and mixing of the two highest energy frontier orbitals -the n + and π localized primarily on the -P=P-unit. A simple, single-configuration model based upon this symmetry-breaking is shown to be consistent with the major features of the measured UV/ vis spectra of several diphosphenes. Detailed evaluation of singlet excitations, transition energies and oscillator strengths with TDDFT showed that the lowest energy transition (S 1 ← S 0 ) does not always correspond to the LUMO ← HOMO configuration. Coupling between the phenyl rings and central -P=P-destabilizes the π-π* dominated state. Hence, the S 1 is always n + -π* in nature, even with a π-type HOMO. This coupling of the ring and -P=P-π systems engenders complexity in the UV/vis absorption region, and may be the origin of the variety of photobehaviours observed in diphosphenes.
2-(4-Aminophenyl)benzothiazoles related to 1 are potentially important pharmaceuticals. Metabolis... more 2-(4-Aminophenyl)benzothiazoles related to 1 are potentially important pharmaceuticals. Metabolism apparently involves oxidation and esterification to 3. In water, hydrolysis and photolysis of 3 generates the nitrenium ion 4 that can be detected indirectly by N(3)(-) trapping and directly by UV-vis spectroscopy following laser flash photolysis. The transient, with lambda(max) 570 nm, and a lifetime of 530 ns, reacts with N(3)(-) at a diffusion-controlled rate and generates the quinol 6 by reaction with water.
The photochemistry of 2-naphthoyl azide was studied in various solvents by femtosecond time-resol... more The photochemistry of 2-naphthoyl azide was studied in various solvents by femtosecond time-resolved transient absorption spectroscopy with IR and UVÀvis detection. The experimental findings were interpreted with the aid of computational studies. Using polar and nonpolar solvents, the formation and decay of the first singlet excited state (S 1 ) was observed by both time-resolved techniques. Three processes are involved in the decay of the S 1 excited state of 2-naphthoyl azide: intersystem crossing, singlet nitrene formation, and isocyanate formation. The lifetime of the S 1 state decreases significantly as the solvent polarity increases. In all solvents studied, isocyanate formation correlates with the decay of the azide S 1 state. Nitrene formation correlates with the decay of the relaxed S 1 state only upon 350 nm excitation (S 0 f S 1 excitation). When S n (n g 2) states are populated upon excitation (λ ex = 270 nm), most nitrene formation takes place within a few picoseconds through the hot S 1 and higher singlet excited states (S n ) of 2-naphthoyl azide. The data correlate with the results of electron density difference calculations that predict nitrene formation from the higher-energy singlet excited states, in addition to the S 1 state. For all of these experiments, no recovery of the ground state was observed up to 3 ns after photolysis, which indicates that both internal conversion and fluorescence have very low efficiencies. 9752 dx.
The photochemistry of three carbonyl azides was studied by ultrafast time-resolved IR spectroscop... more The photochemistry of three carbonyl azides was studied by ultrafast time-resolved IR spectroscopy. Benzoyl, 2-naphthoyl, and pivavoyl azides are promoted to upper excited states S(n) with 270 nm excitation in chloroform. The S(n) states decay in 300 fs to form both the carbonylnitrenes and the S(1) excited states. The decay of the S(1) states of the carbonyl azides correlates with the growth of isocyanates. Formation of carbonylnitrene from S(1) is at most a minor process if it happens at all. The quantum yields of azide decomposition of these azides with 270 nm light are close to unity in chloroform.
Aryloxenium ions 1 are reactive intermediates that are isoelectronic with the better known arylca... more Aryloxenium ions 1 are reactive intermediates that are isoelectronic with the better known arylcarbenium and arylnitrenium ions. They are proposed to be involved in synthetically and industrially useful oxidation reactions of phenols. However, mechanistic studies of these intermediates are limited. Until recently, the lifetimes of these intermediates in solution and their reactivity patterns were unknown. Previously, the quinol esters 2 have been used to generate 1, which were indirectly detected by azide ion trapping to generate azide adducts 4 at the expense of quinols 3, during hydrolysis reactions in the dark. Laser flash photolysis (LFP) of 2b in the presence of O 2 in aqueous solution leads to two reactive intermediates with λ max 360 and 460 nm, respectively, while in pure CH3CN only one species with λmax 350 nm is produced. The intermediate with λ max 460 nm was previously identified as 1b based on direct observation of its decomposition kinetics in the presence of N 3 -, comparison to azide ion trapping results from the hydrolysis reactions, and photolysis reaction products (3b). The agreement between the calculated (B3LYP/6-31G(d)) and observed time-resolved resonance Raman (TR 3 ) spectra of 1b further confirms its identity. The second intermediate with λ max 360 nm (350 nm in CH3CN) has been characterized as the radical 5b, based on its photolytic generation in the less polar CH 3CN and on isolated photolysis reaction products (6b and 7b). Only the radical intermediate 5b is generated by photolysis in CH 3CN, so its UV-vis spectrum, reaction products, and decay kinetics can be investigated in this solvent without interference from 1b. In addition, the radical 5a was generated by LFP of 2a and was identified by comparison to a published UV-vis spectrum of authentic 5a obtained under similar conditions. The similarity of the UV-vis spectra of 5a and 5b, their reaction products, and the kinetics of their decay confirm the assigned structures. The lifetime of 1b in aqueous solution at room temperature is 170 ns. This intermediate decays with firstorder kinetics. The radical intermediate 5b decomposes in a biphasic manner, with lifetimes of 12 and 75 µs. The decay processes of 5a and 5b were successfully modeled with a kinetic scheme that included reversible formation of a dimer. The scheme is similar to the kinetic models applied to describe the decay of other aryloxy radicals.
A series of phosphaalkenes, E-ArP=C(H)Ar&... more A series of phosphaalkenes, E-ArP=C(H)Ar' (Ar = 2,6-Mes2C6H3, Ar' = Ph (1a); Ar = 2,6-Mes2C6H3, Ar' = p-C6H4Br (2a); Ar = 4-Br-2,6-Mes2C6H2, Ar' = Ph (3a); Ar = 4-Br-2,6-Mes2C6H2, Ar' = p-C6H4Br (4a)) have been prepared by phospha-Wittig reactions and characterized. Exposure of these materials either to room light over an extended period of time (days) or to UV light (hours) produced equilibrium mixtures of the E and Z isomers (1b-4b) as indicated by 1H and 31P NMR spectroscopy. The structures of compounds 4a and 4b were determined by single-crystal X-ray diffraction methods. Variable-temperature (1)H NMR studies of 4b indicate hindered rotation about the P-CAr bond, with DeltaH(double dagger) = 13.8 kcal/mol and DeltaS(double dagger) = 1.3 eu. The electronic structures of E- and Z-PhP=C(H)Ph have been examined using density functional theory.
The details of the photophysics of a diphosphene DmpP=PDmp (Dmp: 2,6-Mes(2)C(6)H(3)) have been ex... more The details of the photophysics of a diphosphene DmpP=PDmp (Dmp: 2,6-Mes(2)C(6)H(3)) have been examined experimentally and computationally. Femtosecond transient absorption spectroscopy has been used to probe the dynamics of the S(1) and S(2) excited states of DmpP=PDmp, through excitation at 480 and 400 nm, respectively. The molecule returns to S(0) on sub-nanosecond timescales; no irreversible photochemistry is observed. The S(2) state is observed in the transient spectra with an absorption feature at the red end of the visible spectrum. Its lifetime was measured to be 275 fs. The S(1) state does not absorb appreciably in the probe wavelength range. Excitation into either of these states leads to transient absorption signals in the 400-600 nm region that exhibit a rise time longer than the measured instrument response function, indicating that they do not arise from the initially excited state. These bands decay biexponentially, with lifetimes of ~20 ps and of a few hundred ps. Calculations at the CASSCF(8,6)/6-31G** and CASPT2(8,6)/6-31G**//CASSCF(8,6)/6-31G** levels support these assignments, and underpin an initial working model that involves participation of phenyl torsional twisting motions and the possibility of rapid intersystem crossing to the low-lying triplet manifold.
The journal of physical chemistry. B, Jan 18, 2014
Protein conformational heterogeneity and dynamics are known to play an important role in enzyme c... more Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm(-1), respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases a...
Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicot... more Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD(+)·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C2═O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2═O group and active site residues, as shown by the observation of at least four C2═O stretch bands indicating varying degrees of C2═O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.
Aryl diphosphenes (Ar-P=P-Ar) possess features that may make them useful in photonic devices, inc... more Aryl diphosphenes (Ar-P=P-Ar) possess features that may make them useful in photonic devices, including the possibility for photochemical E-Z isomerization. Development of good models guided by computations is hampered by poor correspondence between predicted and experimental UV/vis absorption spectra. An hypothesis that the phenyl twist angle (i.e. PPCC torsion) accounts for this discrepancy is explored, with positive findings. DFT and TDDFT (B3LYP) were applied to the phenyl-P=P-phenyl (Ph-P=P-Ph) model compound over a range of phenyl twist angles, and to the Ph-P=P-Ph cores of two crystallographically characterized diphosphenes: bis-(2,4,6-tBu 3 C 6 H 2 )diphosphene (Mes*-P=P-Mes*) and bis-(2,6-Mes 2 C 6 H 3 )-diphosphene (Dmp-P=P-Dmp). A shallow PES is observed: the full range of phenyl twist angles is accessible for under 5 kcal/mol. The Kohn-Sham orbitals (KS-MOs) exhibit stabilization and mixing of the two highest energy frontier orbitals -the n + and π localized primarily on the -P=P-unit. A simple, single-configuration model based upon this symmetry-breaking is shown to be consistent with the major features of the measured UV/ vis spectra of several diphosphenes. Detailed evaluation of singlet excitations, transition energies and oscillator strengths with TDDFT showed that the lowest energy transition (S 1 ← S 0 ) does not always correspond to the LUMO ← HOMO configuration. Coupling between the phenyl rings and central -P=P-destabilizes the π-π* dominated state. Hence, the S 1 is always n + -π* in nature, even with a π-type HOMO. This coupling of the ring and -P=P-π systems engenders complexity in the UV/vis absorption region, and may be the origin of the variety of photobehaviours observed in diphosphenes.
2-(4-Aminophenyl)benzothiazoles related to 1 are potentially important pharmaceuticals. Metabolis... more 2-(4-Aminophenyl)benzothiazoles related to 1 are potentially important pharmaceuticals. Metabolism apparently involves oxidation and esterification to 3. In water, hydrolysis and photolysis of 3 generates the nitrenium ion 4 that can be detected indirectly by N(3)(-) trapping and directly by UV-vis spectroscopy following laser flash photolysis. The transient, with lambda(max) 570 nm, and a lifetime of 530 ns, reacts with N(3)(-) at a diffusion-controlled rate and generates the quinol 6 by reaction with water.
The photochemistry of 2-naphthoyl azide was studied in various solvents by femtosecond time-resol... more The photochemistry of 2-naphthoyl azide was studied in various solvents by femtosecond time-resolved transient absorption spectroscopy with IR and UVÀvis detection. The experimental findings were interpreted with the aid of computational studies. Using polar and nonpolar solvents, the formation and decay of the first singlet excited state (S 1 ) was observed by both time-resolved techniques. Three processes are involved in the decay of the S 1 excited state of 2-naphthoyl azide: intersystem crossing, singlet nitrene formation, and isocyanate formation. The lifetime of the S 1 state decreases significantly as the solvent polarity increases. In all solvents studied, isocyanate formation correlates with the decay of the azide S 1 state. Nitrene formation correlates with the decay of the relaxed S 1 state only upon 350 nm excitation (S 0 f S 1 excitation). When S n (n g 2) states are populated upon excitation (λ ex = 270 nm), most nitrene formation takes place within a few picoseconds through the hot S 1 and higher singlet excited states (S n ) of 2-naphthoyl azide. The data correlate with the results of electron density difference calculations that predict nitrene formation from the higher-energy singlet excited states, in addition to the S 1 state. For all of these experiments, no recovery of the ground state was observed up to 3 ns after photolysis, which indicates that both internal conversion and fluorescence have very low efficiencies. 9752 dx.
The photochemistry of three carbonyl azides was studied by ultrafast time-resolved IR spectroscop... more The photochemistry of three carbonyl azides was studied by ultrafast time-resolved IR spectroscopy. Benzoyl, 2-naphthoyl, and pivavoyl azides are promoted to upper excited states S(n) with 270 nm excitation in chloroform. The S(n) states decay in 300 fs to form both the carbonylnitrenes and the S(1) excited states. The decay of the S(1) states of the carbonyl azides correlates with the growth of isocyanates. Formation of carbonylnitrene from S(1) is at most a minor process if it happens at all. The quantum yields of azide decomposition of these azides with 270 nm light are close to unity in chloroform.
Aryloxenium ions 1 are reactive intermediates that are isoelectronic with the better known arylca... more Aryloxenium ions 1 are reactive intermediates that are isoelectronic with the better known arylcarbenium and arylnitrenium ions. They are proposed to be involved in synthetically and industrially useful oxidation reactions of phenols. However, mechanistic studies of these intermediates are limited. Until recently, the lifetimes of these intermediates in solution and their reactivity patterns were unknown. Previously, the quinol esters 2 have been used to generate 1, which were indirectly detected by azide ion trapping to generate azide adducts 4 at the expense of quinols 3, during hydrolysis reactions in the dark. Laser flash photolysis (LFP) of 2b in the presence of O 2 in aqueous solution leads to two reactive intermediates with λ max 360 and 460 nm, respectively, while in pure CH3CN only one species with λmax 350 nm is produced. The intermediate with λ max 460 nm was previously identified as 1b based on direct observation of its decomposition kinetics in the presence of N 3 -, comparison to azide ion trapping results from the hydrolysis reactions, and photolysis reaction products (3b). The agreement between the calculated (B3LYP/6-31G(d)) and observed time-resolved resonance Raman (TR 3 ) spectra of 1b further confirms its identity. The second intermediate with λ max 360 nm (350 nm in CH3CN) has been characterized as the radical 5b, based on its photolytic generation in the less polar CH 3CN and on isolated photolysis reaction products (6b and 7b). Only the radical intermediate 5b is generated by photolysis in CH 3CN, so its UV-vis spectrum, reaction products, and decay kinetics can be investigated in this solvent without interference from 1b. In addition, the radical 5a was generated by LFP of 2a and was identified by comparison to a published UV-vis spectrum of authentic 5a obtained under similar conditions. The similarity of the UV-vis spectra of 5a and 5b, their reaction products, and the kinetics of their decay confirm the assigned structures. The lifetime of 1b in aqueous solution at room temperature is 170 ns. This intermediate decays with firstorder kinetics. The radical intermediate 5b decomposes in a biphasic manner, with lifetimes of 12 and 75 µs. The decay processes of 5a and 5b were successfully modeled with a kinetic scheme that included reversible formation of a dimer. The scheme is similar to the kinetic models applied to describe the decay of other aryloxy radicals.
A series of phosphaalkenes, E-ArP=C(H)Ar&... more A series of phosphaalkenes, E-ArP=C(H)Ar' (Ar = 2,6-Mes2C6H3, Ar' = Ph (1a); Ar = 2,6-Mes2C6H3, Ar' = p-C6H4Br (2a); Ar = 4-Br-2,6-Mes2C6H2, Ar' = Ph (3a); Ar = 4-Br-2,6-Mes2C6H2, Ar' = p-C6H4Br (4a)) have been prepared by phospha-Wittig reactions and characterized. Exposure of these materials either to room light over an extended period of time (days) or to UV light (hours) produced equilibrium mixtures of the E and Z isomers (1b-4b) as indicated by 1H and 31P NMR spectroscopy. The structures of compounds 4a and 4b were determined by single-crystal X-ray diffraction methods. Variable-temperature (1)H NMR studies of 4b indicate hindered rotation about the P-CAr bond, with DeltaH(double dagger) = 13.8 kcal/mol and DeltaS(double dagger) = 1.3 eu. The electronic structures of E- and Z-PhP=C(H)Ph have been examined using density functional theory.
The details of the photophysics of a diphosphene DmpP=PDmp (Dmp: 2,6-Mes(2)C(6)H(3)) have been ex... more The details of the photophysics of a diphosphene DmpP=PDmp (Dmp: 2,6-Mes(2)C(6)H(3)) have been examined experimentally and computationally. Femtosecond transient absorption spectroscopy has been used to probe the dynamics of the S(1) and S(2) excited states of DmpP=PDmp, through excitation at 480 and 400 nm, respectively. The molecule returns to S(0) on sub-nanosecond timescales; no irreversible photochemistry is observed. The S(2) state is observed in the transient spectra with an absorption feature at the red end of the visible spectrum. Its lifetime was measured to be 275 fs. The S(1) state does not absorb appreciably in the probe wavelength range. Excitation into either of these states leads to transient absorption signals in the 400-600 nm region that exhibit a rise time longer than the measured instrument response function, indicating that they do not arise from the initially excited state. These bands decay biexponentially, with lifetimes of ~20 ps and of a few hundred ps. Calculations at the CASSCF(8,6)/6-31G** and CASPT2(8,6)/6-31G**//CASSCF(8,6)/6-31G** levels support these assignments, and underpin an initial working model that involves participation of phenyl torsional twisting motions and the possibility of rapid intersystem crossing to the low-lying triplet manifold.
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