Stability of Flavin Semiquinones in the Gas Phase: The Electron

Sep 5, 2013 - The anions were formed by attachment of scattered electrons trapped in the ICR cell. After a variable delay, an anionic spectrum was obt...
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Stability of Flavin Semiquinones in the Gas Phase: The Electron Affinity, Proton Affinity, and Hydrogen Atom Affinity of Lumiflavin Tianlan Zhang, Kaitlin Papson, Richard Ochran, and Douglas P. Ridge* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *

ABSTRACT: Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formylphenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol−1. Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.

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INTRODUCTION Flavin radical species, although first observed in 1936 by Michaelis and co-workers,1,2 still occupy the attention of enzymologists.3 For example, studies have examined the reactions of radical flavin semiquinones with cytochromes4−6 and the occurrence of unusually stable flavin semiquinones.7−9 The stability of the radical flavin semiquinones is central to the unique biological function of flavin enzymes. Having stable radical forms, the flavins function in both one electron and two electron oxidation processes. Therefore, they can effectively couple a two electron donor such as NADH to one electron acceptors such as the cytochromes.10 Scheme 1 shows flavin structures, in three oxidation states: oxidized, semiquinone, and fully reduced. The present study was initiated to provide a measurement of the intrinsic stability of the semiquinone forms of flavin. In particular the study includes measurements of the gas phase electron affinity of an oxidized flavin and the proton affinity of the resulting radical anion. The electron affinity of the flavin and the proton affinity of the radical ion can be combined to give the hydrogen atom affinity of the oxidized flavin. These values are then compared with the free energy for reducing flavin to a semiquinone deduced from condensed phase potentiometric measurements.11,12 The measurement of the gas phase electron affinity used the methods of Fourier transform ion cyclotron resonance mass spectrometry (FT© 2013 American Chemical Society

Scheme 1. Structures of Flavin in Various Oxidation States

Received: July 9, 2013 Revised: September 4, 2013 Published: September 5, 2013 11136

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ICR-MS) similar to those used to measure electron affinities of metalloporphyrins.13 The electron affinity measurements depend on the scale of gas phase electron affinities established by Kebarle and co-workers.14,15 The proton affinity measurement also used FT-ICR-MS methods and depends on the scale of gas phase acidities established by a number of workers.16 Also reported here is the proton affinity of the oxidized flavin determined using FT-ICR-MS methods to follow the gas phase proton transfer reactions of the flavin with various proton donors. The proton affinity is useful in evaluating the basicity of the various sites in the molecule and hence its interactions with solvents and enzymes. The flavins differ in their substitution on the nitrogen at position 10 of isoalloxazine ring system (R in Scheme 1). Lumiflavin has a methyl substituent at position 10, while riboflavin (vitamin B 2 ) has a ribitol substituent (−CH2(CHOH)3CH2OH) at the 10 position. Other side chains are ribitol phosphate (FMN) and ribityl adenine diphosphate (FAD). Since the isoalloxazine ring system is the redox active part of all of these molecules and since lumiflavin is somewhat more volatile than riboflavin, lumiflavin was chosen for the study. Density functional theory calculations on lumiflavin and its semiquinone forms have been reported.17 Comprehensive theoretical treatments of the condensed phase reduction of lumiflavins have also been reported.18 Additional calculations will be described here, and the theoretical results will be compared to the experimental measurements.

Following the 10 ms pulse of a nominal 2 eV and 1 μA electron beam, the radical parent ions of lumiflavin and 1,4naphthoquinone were formed by attachment of scattered electrons caught in the trap. After 2 s delay to allow completion of electron attachment processes, all the ions were ejected except lumiflavin radical anion. As shown in Figure 1, the

METHODS Experiments were performed using a commercial Fourier transform ion cyclotron resonance mass spectrometer (Extrel FTMS 2000, Madison, WI) equipped with 3 T superconducting magnet, an automatic heatable probe assembly, and batch inlet system. Riboflavin and lumiflavin were introduced into the FT-ICR cell on the heated probe. Positive ion spectra were obtained using a 5 ms pulse of a 70 eV electron beam to produce ions followed by broadband excitation and acquisition of the cyclotron resonance mass spectrum. For the negative ion spectrum, a 2 eV, 1 μA electron beam was pulsed into the cell for 10 ms. The anions were formed by attachment of scattered electrons trapped in the ICR cell. After a variable delay, an anionic spectrum was obtained. The total signal initially increased with delay time reaching a constant value as the electron attachment process went to completion. As a photodegradation product lumiflavin can be observed as an impurity in the riboflavin sample and since it is more volatile than riboflavin, it is readily distilled from the riboflavin as it is heated on the probe. Both negative ion spectra and low energy electron impact positive ion spectra showed the lumiflavin parent ion appearing at a nominal probe temperature of 260 °C. Riboflavin appeared only at 350 °C. The lumiflavin could be observed in the mass spectrum even though the ionization gauge indicated no discernible pressure above background (∼0.1 × 10−8 Torr). The gauge is far enough from the source that the involatile lumiflavin was absorbed on the wall before it reached the gauge. The mass spectrum, however, provided a measure of the relative pressure of lumiflavin in the cell. In a typical electron affinity measurement, both lumiflavin from the heated probe at a temperature of 260 °C and 1,4naphthoquinone from the batch inlet system were admitted into the ICR cell. The nominal pressure was 1.7 × 10−8 Torr.

further reversible electron transfer between the lumiflavin radical anion and the neutral 1,4-naphthoquinone was allowed to come to steady state. The relative steady-state peak intensities were taken to be proportional to the relative concentrations of the ions at equilibrium. Similarly for all equilibrium experiments, the relative intensities of the peaks of the two ionic reactants in the FTICR spectra at steady state were taken as proportional to their relative concentrations. The ratio of neutral pressures was estimated from the electron impact mass spectrum of the mixture and relative ionization cross-sections of the neutrals estimated by the method of Georgiadis and Bartmess.19 This method relates the ionization cross-section to the polarizability. Polarizabilities were estimated using the method of Miller and Savchick.20 To examine proton transfer to the lumiflavin radical anion, the proton donor was introduced into the ICR cell through the batch inlet to react with the isolated lumiflavin radical anion, which was prepared as described above. The nominal pressures were in the 10−8 Torr range. The spectrum was recorded at equal time intervals until the proton-transfer reaction was complete. Density functional calculations were completed using Gaussian03.21 Calculations were done to find the relative stability of oxidized lumiflavin protonated at various sites. The theoretical calculations were also used to explore an alternative pathway from oxidized flavin to neutral semiquinone: adding the H+ first and then the e−, rather than the other way around. A B3LYP functional was used with a 6-31G* basis set.22,23 Optimization-frequency calculations were used, and then structures were optimized until a minimum with zero imaginary frequencies was found. Tight convergence criteria were requested for all calculations.

Figure 1. Variation with time of lumiflavin radical anion and 1,4naphthoquinone anion in a mixture of the two neutrals. Closed circles represent the lumiflavin anion and the closed squares the naphthoquinone.

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efficiencies for the reactions of several other acids with Lfl•−. The rate constants and reaction efficiencies for proton transfer drop by 2 orders of magnitude when the gas phase proton affinity (PA) of the conjugate base of the proton donor increases from 331.0 kcal mol−1 (PA(difluoroacetate)) to 333.0 kcal mol−1 (PA(p-formyl-phenoxide)).27,28 The rate constant drops to unmeasurable at still higher proton affinities. Assuming that reactions with rate constants no less than an order of magnitude less than the collision rate are exothermic or thermoneutral and that slower reactions are endothermic, we conclude that the gas phase proton affinity of Lfl•− (ΔH for Lfl• → Lfl•− + H+) is between 331.0 and 333.0 kcal mol−1 or 332.0 ± 1.0 kcal mol−1, or including the uncertainty associated with proton affinities of the reference acids 332.0 ± 2.2 kcal mol−1. The entropy of deprotonation of the reference acids is almost constant corresponding to the entropy of a free proton. If we assume that lumiflavin follows the behavior of reference bases in this respect, then the gas acidity (GA) of LflH• (ΔG for LflH • → Lfl •− + H + ) is in the range from 323.8 (GA(difluoroacetate)) to 326.1 kcal mol−1 (GA(p-formylphenoxide)) or 325.0 ± 2.2 kcal mol−1 including the uncertainty associated with the proton affinities of the reference acids. Bond Strength of Lumiflavin-H. The bond strength between a hydrogen atom and a lumiflavin molecule can be calculated from the electron affinity of Lfl, EA(Lfl), and the proton affinity of Lfl•−, PA(Lfl•−). The thermodynamic cycle and the thermochemical data required are given in Table 2. Table 2 gives ΔG(reaction 2.6), −52.1 kcal mol−1, in addition to ΔH(reaction 2.6), −59.7 kcal mol−1, for reaction 2.6 in which a bond is formed between gas phase lumiflavin and a H atom to give gas phase lumiflavin semiquinone, LflH•(g). This facilitates the comparison with numbers derived from redox potential measurements shown in Table 3. Table 3 gives the thermodynamic cycle and thermochemical data required to deduce ΔG(reaction 3.5), the free energy for reaction 3.5, the formation of a bond between aqueous lumiflavin,and an H atom to form aqueous LflH•. The crucial number in the cycle outlined in Table 3 is the potential of the Lfl/LflH• couple. The table gives the results of a relatively recent measurement obtained using pulsed radiolysis to form radical anions, which are equilibrated by charge exchange reactions.12 The pulsed radiolysis number is an average result for riboflavin, FMN and FAD, which differ by less than the indicated error. We expect lumiflavin to have this same reduction potential. The only difference between the number derived from gas phase data (ΔG(reaction 2.6)) and that derived from electrochemical data (ΔG(reaction 3.5)) is that the former pertains to gas phase Lfl and LflH•, while the latter pertains to aqueous Lfl and LflH•. A theoretical value of ΔG(reaction 3.5) can be deduced from results calculated using the M06-L density functional, which give ΔGo298(reaction 3.1) as −99 kcal mol−118 and the solvation free energy of the proton as 265.9 kcal mol−1.31 Combining these numbers with the ionization energy of H•(g) gives a theoretical ΔG(reaction 3.5) as −51.3 kcal mol−1 in good agreement with the number in Table 3 from experimental electrochemical measurements, −51.0 ± 0.2 kcal mol−1. The difference between ΔG (reaction 2.6) and ΔG (reaction 3.5) is the difference between the solvation energy of Lfl and that of LflH• or the difference between ΔG(eq 3) and ΔG(eq 4).

RESULTS AND DISCUSSION Electron Affinity of Lumiflavin. Lumiflavin radical anion (Lfl•−), which is a one electron reduced flavin or a flavosemiquinone, can be made either by thermal electron attachment to the lumiflavin (Lfl) molecule or by electron transfer from another species to Lfl. By choosing 1,4naphthoquinone (NpQ), that has an electron affinity of 1.81 ± 0.1 eV, as the reference,15 the electron affinity of Lfl can be determined by measuring the equilibrium constant for the electron-transfer reaction 1 below. Lfl•− + NpQ ↔ Lfl + NpQ•−

(1)

Figure 1 shows a typical variation with time of the relative ion signals of Lfl•− and NpQ•− in a mixture of the two neutrals following ejection of NpQ•− from the trap. The reversible electron transfer eventually reaches equilibrium at long reaction time. The equilibrium constant for reaction 1 deduced from the data in Figure 1 gives ΔG(1) = 0.05 eV. Assuming that ΔG = ΔH for reaction 1, we deduce that EA(Lfl) = 1.86 ± 0.1 eV. The error estimate for the EA value comes from the estimated uncertainty in the electron affinity scale (±0.1 eV). The estimated error in the measured value of the equilibrium constant for reaction 1 is ±40% corresponding to an uncertainty in the EA of ±0.01 eV. Proton Affinity of Lumiflavin Radical Anion. A neutral semiquinone form of lumiflavin (LflH•) can be made by protonating its radical anion species. The proton affinity of Lfl•− was determined using the bracketing method. The Lfl•− ion was formed by thermal electron attachment and then allowed to react with different neutral proton donors in the ICR cell. Reaction 2 was observed. Lfl•− + F2CHCO2 H → LflH•+F2CHCO2−

(2)

For reaction 2, the relative ion intensities of two ionic species verses reaction time are plotted in Figure 2. The proton transfer

Figure 2. Kinetic spectrum of the proton transfer from difluoroacetic acid to lumiflavin anion.

from difluoroacetic acid to Lfl•− goes to completion in about 180 s. Analysis of the data in Figure 2 together with an estimate of the pressure using the method of Georgiadis and Bartmess19 gives a rate constant for reaction 2 of 2.1 × 10−10 cm3 molecule−1 s−1 as indicated in Table 1. This corresponds to an efficiency of 0.17 (efficiency = k/kc, kc = capture collision rate calculated using the method of Su and Chesnavich24−26). Table 1 also lists the similarly determined rate constants and 11138

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Table 1. Rate Constants of Proton Transfer Reaction of HX with Lumiflavin ΔGacida

HX HBr Cl2CHCO2H F2CHCO2H p-CHO(C6H4)OH HCl

318.2 321.9 323.8 326.1 328.0

± ± ± ± ±

ΔHacidb

2.0 2.0 2.0 2.0 2.0

323.5 328.4 331.0 333.0 333.4

± ± ± ± ±

2.1 2.0 2.0 2.0 2.0

k × 1010c

k/kcd

4.0 2.3 2.1 0.017