Electronic Transition Dipole Moment Directions of Reduced Anionic

The IR and UV/vis linear dichroic spectra of reduced anionic flavin mononucleotide (FMNH-) partially oriented in poly(vinyl alcohol) (PVA) films have ...
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J. Phys. Chem. B 2008, 112, 119-126

119

Electronic Transition Dipole Moment Directions of Reduced Anionic Flavin in Stretched Poly(vinyl alcohol) Films M. Salim U. Siddiqui,† Goutham Kodali,‡ and Robert J. Stanley*,†,‡ Department of Biochemistry and Department of Chemistry, Temple UniVersity, Philadelphia, PennsylVania 19122 ReceiVed: July 24, 2007; In Final Form: September 27, 2007

The IR and UV/vis linear dichroic spectra of reduced anionic flavin mononucleotide (FMNH-) partially oriented in poly(vinyl alcohol) (PVA) films have been measured to determine the direction of the major electronic transition dipole moments. The IR linear dichroism (LD) was measured in the 1750-1350 cm-1 region to provide the overall molecular orientation of the FMNH- in the stretched films. Time-dependent density functional theory using the B3LYP functional was used to calculate the normal modes and the transition dipole moments of reduced lumiflavin. The calculated normal modes assisted in IR band assignments and in the determination of the IR transition dipole moment directions which were required for the determination of the orientation parameters for FMNH- in PVA films. The UV/vis LD spectrum was measured over the 200700 nm region and was resolved into contributions from three π f π* transitions. The directions of the transitions are 90° ( 4° at 440 nm, 79° ( 4° at 350 nm, and 93° ( 4° at 290 nm with counterclockwise rotations with respect to the N5-N10 axis. Comparison of the calculated and experimentally determined transition dipole moments allowed for refined assignment of the transition dipole moment directions. To our knowledge, this is the first experimental evidence that the 350-450 nm absorption arises from two unique transitions. Remarkably, the two lowest energy transition dipole moments for FMNH- are nearly parallel to those obtained in prior studies for both oxidized and semiquinone flavin.

Introduction The flavin molecule is a complex heterocyclic redox reagent that can participate in both one-electron- and two-electrontransfer reactions.1-5 As a result of this unique redox ability, flavins function as coenzymes throughout a wide range of biological redox reactions. Flavins possess three oxidation states: oxidized flavoquinone, one-electron reduced flavosemiquinone radical, and two-electron reduced flavohydroquinone. Each of these oxidation states has been investigated through several theoretical and experimental studies.1 Optical spectroscopy of flavins and flavoproteins6 has played a vital role in understanding and characterizing the ground-state and excitedstate electron transfer of flavins in biological processes.7-11 The interpretation of some optical spectroscopy studies requires a knowledge of the fundamental electronic and photochemical properties of flavins. For example, the application and interpretation of various polarization-based spectroscopic techniques (e.g., FRET, CD) to flavoproteins require a knowledge of the transition dipole moment directions of flavins. While studies have determined the direction of the electronic transition dipole moments of the neutral oxidized and the semiquinone states of flavins12-14, the experimental determination of the electronic transition dipole moment directions of reduced anionic flavins has not yet appeared in the literature. As a result, polarization-based spectroscopic techniques have been utilized in only a limited capacity for reduced flavoproteins. One method to determine the electronic transition dipole moment direction is UV/vis linear dichroism (UV/vis-LD) * Corresponding author. E-mail: [email protected]. Phone: (215) 204-2027. Fax: (215) 204-1532. † Department of Biochemistry. ‡ Department of Chemistry.

spectroscopy on reduced flavin samples partially oriented in stretched poly(vinyl alcohol) (PVA) films. This technique has been successfully employed to study oxidized flavins12 and many other molecules.15 The primary problem in analyzing results from UV/vis-LD spectroscopy arises from the difficulty in establishing the molecular orientation of a molecule with low symmetry within the stretched PVA film. This difficulty can be overcome by measuring the IR linear dichroic (IR-LD) spectrum of the sample in the same stretched PVA films. Under ideal circumstances, the analysis of the IR-LD spectrum establishes a preferred molecular orientation axis and a molecular coordinate system in the stretched PVA films. Using the molecular orientation axis established from the IR-LD spectrum, an analysis of the UV/vis-LD spectrum provides the directions of the electronic transition dipole moments relative to this axis. Together, the IR-LD and UV/vis-LD studies establish the directions of the electronic transition dipole moments within the molecular coordinate system. This report presents the first IR and UV/vis linear dichroic spectra of partially oriented, reduced anionic flavin mononucleotide (FMNH-, see Scheme 1) in stretched PVA films. The analysis does not assume any particular molecular orientation or shape but rather starts from a general expression for the linear dichroism. The IR-LD experiments provide information about the molecular orientation of FMNH- in the stretched PVA films. In addition, time-dependent density functional theory (TDDFT) is used to calculate the normal modes and the electronic transition dipole moments of reduced lumiflavin. The calculated normal modes allow for IR band assignments and the determination of the IR transition moment directions. The IR transition moment directions are required for the determination of the orientation parameters for the FMNH- in the PVA film, which

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120 J. Phys. Chem. B, Vol. 112, No. 1, 2008 SCHEME 1: FMNH- Structure and Numbering

are applied to the analysis of the UV/vis-LD studies and hence the determination of the electronic transition dipole moments.15 Additionally, comparison of the calculated results with the experimental results allows for the refined assignment of the directions of the major electronic transition dipole moments of FMNH- from the UV/vis linear dichroic experiments. Our work reveals that the 350-450 nm region of the FMNH- absorption spectrum is due to two bands of differing LD values. This observation is contrary to the commonly held assumption that this absorption is due to a single transition centered at ∼350 nm. Methods and Materials Materials. Elavanol 71-30 (PVA powder) was a generous gift from Mr. Elliott Echt of the Dupont Corporation. Flavin mononucleotide (FMN, 95% purity) was purchased from Sigma and used without further purification. Film Preparation. The sample and reference films were prepared in a MACS anaerobic workstation (Microbiology International) by mixing the deaerated PVA powder in the appropriate deaerated sample or reference solution. Except for the collection of the LD spectra, subsequent handling of both sample and reference films was done within the MACS anaerobic workstation. In sample films, the reduced flavin mononucleotide (0.3 mM to 1.2 mM solution) was mixed into the PVA solution prior to heating. The reference solution was prepared and handled in the same manner as the sample solution, except that it did not contain FMN. Typically, a 10% (w/w) PVA solution was used. The sample and reference film solutions were heated for 30 min in a silicone oil bath at 85-95 °C with continuous stirring. During heating, the solutions were covered by glass bottle stoppers to prevent the formation of thick PVA skins on the solution surface. Approximately 15 min into heating, 5% (v/v) triethylamine (Fisher) was added to the film solution to generate anionic reduced flavin. Without the addition of triethylamine, both anionic and neutral species of reduced flavin are formed in the PVA film. The resulting “mixed” flavin films proved difficult to analyze unambiguously. After heating, the solutions were allowed to cool for 10 min at room temperature within the MACS anaerobic chamber. The solutions were then carefully poured in a dropwise fashion onto horizontal microscope slides. The film thickness varied between 40 µm and 100 µm for various reduced flavin concentrations. The poured films polymerized over 16-20 h inside the anaerobic chamber. All film preparation steps were performed under yellow light conditions to prevent undesired photochemistry. Reduced Anionic Flavin Mononucleotide (FMNH-). FMN was dissolved in deaerated distilled water containing EDTA (Fisher). EDTA was used as an electron donor for photoreduction. While various amounts of EDTA were tested, a 15:1 mole ratio of EDTA/FMN was sufficient for flavin photoreduction and subsequent film preparation. The FMN-EDTA solution was sealed in a Pyrex round-bottom flask with an airtight turnoverseptum rubber stopper (Fisher). The FMN-EDTA solution flask was then placed in a water bath, continuously stirred, and

Siddiqui et al. irradiated with filtered (WG 340 filter) 150 W Xe arc lamp (Hamamatsu) to photo reduce the FMN. The photoreduction progress was monitored by the loss of the intense fluorescence of the oxidized FMN when irradiated with 365 nm light from a ENF-240C hand-held UV lamp (Spectraline). The complete photoreduction of a 25 mL stock solution required ∼3 h of irradiation at a distance of ∼5 cm from the output lens of the lamp. Photoreduction was confirmed by UV/vis spectroscopy on a HP8452 UV/vis spectrophotometer. Another possible complication arises from protonation of N1 within the PVA films. The pKa of the N1-H is ∼6.7 with a neutral reduced flavin species existing at pH below this pKa. Several attempts were made to adjust the pH within the PVA matrix by adjusting the pH of the starting reduced flavin solution. Various buffers were tried, but only free base EDTA afforded reduced flavin in an optically pristine PVA film. Many of the other buffers would salt out of the PVA film during the polymerization process. However, EDTA alone produced films in which both anionic and neutral species were present in approximately equal proportions. However, the addition of 5% (v/v) triethylamine afforded a >99% anionic reduced flavin. Further increases in the amount of triethylamine did not improve the percentage of anionic reduced flavin but rather resulted in PVA films of poor optical quality. Film Stretching. All manipulations of the PVA films were performed in the MACS anaerobic chamber. After polymerization, the films were carefully removed from the microscope slides with a razor blade, a spatula, or both. Both sample and reference films were stretched on a homemade mechanical stretching device consisting of two optical translation stages (Newport 423) connected together on a base plate. The films were clamped between the two translation stages. The clamping plates were carefully tightened down to minimize ripple formation in the films. After the films were loaded, the stretching device was placed under an infrared heating lamp (Infrared Internationale) in the anaerobic chamber. The voltage supplied to the heating lamp was passed through a voltage regulator (Variac) to control the temperature of the heating lamp. The heating lamp and stretching device were allowed to equilibrate to ∼90 °C over an hour. The stretching of the polymer films was performed by carefully turning the micrometers on the translation stages. The optical quality of the stretched film was highly sensitive to temperature control during the stretching process. As a result, films were stretched in intervals: periods of stretching followed by periods of equilibration to ∼90 °C. The films were stretched over a period of 3-6 h depending upon the desired stretch factor. The stretch factor (Fs) is defined as the ratio of the final stretched length to the original length before stretching. IR and UV/Vis Spectroscopy. The IR and UV/vis linear dichroic spectra were measured on a Nicolet 560 FTIR spectrophotometer and a HP 8452A diode array UV/vis spectrophotometer, respectively. The spectrophotometers were fitted with special homemade mountable platforms. The general design of the platforms was the same for both spectrophotometers: a rotatable polarizer mounted between the rotatable film stage and the light source. The polarizer was set to allow maximal transmission of the lamp light for both IR and UV/vis studies. Additionally, in both cases, the polarizer was calibrated by measuring transmittances through the polarizer at different angles in the absence of a film in the rotatable film stage. A SPECAC IGP228 polarizer (CVI Laser, LLC) was used for the IR spectrophotometer. A calcite polarizer was used for the UV/ vis spectrophotometer.

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Stretched films were loaded between two CaF2 windows (Pike Technologies) for the IR studies and two UV fused-silica windows (Edmund Industrial Optics) for the UV/vis studies. In both cases, the films were trimmed to fit the circular profile of the windows. The film-window sandwich was loaded into a Newport rotatable stage such that the stretching axis (i.e., Z axis) was perpendicular to the floor of the laboratory. This rotatable film stage position was set as 0°, and rotations were measured from this position. The film was rotated and absorbances were measured in intervals of 10° from 0° to 180°. The absorbances of the reference and sample films were measured separately for the UV/vis studies. Subtracting the reference spectra from the corresponding sample spectra generated the reference-corrected UV/vis absorbance spectra. For the IR studies, the transmittance of the reference and sample films was measured, and the logarithmic ratio of the reference spectra to the corresponding sample spectra provided the IR absorbance spectra. Basic Equations for LD Analysis. The reduced linear dichroism (LDr) can be simply defined as:

LDr )

LD A| - A⊥ ) Aiso Aiso

(1)

where A| and A⊥ represent the corrected dichroic absorbances with light polarized parallel and perpendicular to the stretching axis (i.e., Z axis), respectively, and Aiso represents the isotropic absorbance of the sample.15,16 Normalization with Aiso removes any dependence upon path length, concentration, and the dipole strength of the transition in the LD spectra. Therefore, the LDr is only a function of the direction of the transition dipole moment relative to the polarization of the probe light. For a low-symmetry molecule, such as FMNH-, the distribution of reduced flavin molecules is uniform along the stretching Z axis and the orientation is characterized by a nondiagonal orientation tensor:15,16

[

]

Sx′x′ Sx′y′ Sx′z′ S′ ) Sy′x′ Sy′y′ Sy′z′ ) Sz′x′ Sz′y′ Sz′z′

[

1 (3 cos2 ξ′ - 1) 3 cos ξ′ cos ψ′ 3 cos ξ′ cos ζ′ 2 1 (3 cos2 ψ′ - 1) 3 cos ζ′ cos ψ′ 3 cos ξ′ cos ψ′ 2 1 (3 cos2 ζ′ - 1) 3 cos ξ′ cos ζ′ 3 cos ζ′ cos ψ′ 2

]

(2)

where, in an arbitrarily selected orthogonal x′y′z′ coordinate system within the molecular framework, ξ′, ψ′, and ζ′ are the angles between the x′, y′, and z′ axes and Z axis, respectively. If the reduced flavin molecule is assumed to be relatively planar, then the orientation process (i.e., the stretching of the PVA film) should result in the flavin molecules presenting a minimum cross-sectional area to the Z axis. In other words, an orientation axis exists within the molecular frame that is parallel to the Z axis, and all angles of rotation around this axis are equally probable.15,16 The x′ axis is defined to be perpendicular to the molecular plane of the reduced flavin, and y′ and z′ axes lie in the molecular plane. In this case, only transitions with transition dipole moments in the molecular plane are observed, and the reduced dichroism, in the x′y′z′ axis system, can be expressed as

LDr ) 3(Sy′y′ sin2 θ′ + Sz′z′ cos2 θ′ + Sy′z′ sin θ′ cos θ′)

(3)

where θ′ is the angle between the transition dipole moment and the z′ axis, and Sy′y′, Sz′z′, and Sy′z′ are order parameters:

1 Sy′y′ ) (3 cos2 ψ′ - 1) 2 1 Sz′z′ ) (3 cos2 ζ′ - 1) 2 Sy′z′ ) 3 cos ζ′ cos ψ′

(4)

These three orientation parameters represent three unknowns. If the x′y′z′ axis system is rotated by an angle R about the x′ axis into a new xyz axis system, eq 3 becomes:

LDr ) 3(Syy sin2 θ + Szz cos2 θ + Syz sin θ cos θ)

(5)

where θ ) θ′ - R is the angle between the transition dipole moment and the z axis. Moreover, an angle R0 exists such that Syz ) 0 and the orientation tensor diagonalizes with Szz and Syy becoming extrema. In the diagonal xyz axis system, the order parameters are labeled to fulfill Szz > Syy and Sxx + Syy + Szz ) 0. These diagonal order parameters and the angle R0 are given by the following equations:

1 Syy ) Sy′y′ cos2 R0 + Sz′z′ sin2 R0 - Sy′z′ sin 2R0 2 1 Szz ) Sy′y′ sin2 R0 + Sz′z′ cos2 R0 + Sy′z′ sin 2R0 2 tan 2R0 ) Sy′z′/(Sz′z′ - Sy′y′)

(6)

In the diagonal xyz axis system, eq 5 reduces to:

LDr ) 3(Syy sin2 θ + Szz cos2 θ)

(7)

Although other formalisms exist, analysis of LDr spectra can be achieved using the above equations.12 Computational Methods. All calculations were performed on a UNIX system using the Gaussian 03, revision C.01 package of ab initio programs.17 The methods employed include conventional ab initio procedures (i.e., RHF/6-31G* and RHF/631G**) and density functional theory (DFT) procedures (i.e., B3-LYP/6-31G* and B3-LYP/6-31G**). As in previous studies18-21 and because of computational constraints, lumiflavin was used as a model compound for the calculation of the normal modes and the transition dipole moments of reduced flavin. The coordinates of the heavy atoms of lumiflavin used for the calculations were obtained from the coordinates of the isoalloxazine moiety of the flavin cofactor in the crystal structure of DNA photolyase bound to a CPDlike substrate (Protein Data Bank accession number 1TEZ).22 Hydrogens were inserted using appropriate bond lengths and angles. This initial geometry of the reduced lumiflavin was optimized at the RHF/6-31G* level of theory. The calculation of the transition dipole moments and the normal modes used the DFT procedures (i.e., B3-LYP with a basis set of 6-31G* and/or 6-31G**). Results and Discussion IR Absorption Spectra of FMNH- in PVA Films. Before the directions of the electronic transition dipole moments of the reduced anionic flavin can be determined within the

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Siddiqui et al. TABLE 1: Wavenumbers (cm-1) and Assignments of FMNH- IR Bands (1750 to 1350 cm-1) experimental calculated energy (cm-1) energya (cm-1) 1672 1623 1593b 1573

assignment

missing 1630 (1752) 1597 (1718)

C2dO stretch C4dO stretch C4dO stretch and N3sH bend

1544 (1660)

C4adC10a, C6sH, C9sH bend

1528 (1643) 1494 (1612)

C4adC10a, C6sH, and C9sH bend C6sH, C9sH bend, and N5sH bend

1463 (1574) 1439 (1547)

C6sH, C9sH bend, and N5sH bend ring-I mode and N5sH bend

1434 (1542) 1425 (1532) 1390 (1494) 1353 (1454)

C6sH, C9sH bend, and N5sH bend ring-I mode ring-I mode and N5sH bend ring-I mode, C9sH, and N5sH bend

1537 1500b 1472 1439b 1413

Figure 1. IR absorption spectrum with fit and computationally determined frequencies of FMNH-. The IR absorption spectrum of FMNH- in PVA films (O) with an minimal deconvolution into 11 Gaussian bands (s). The sum of the 11 bands is represented by the line coincident with the - symbol. The TDDFT-computed frequencies (9 with drop line) are scaled and plotted for comparison.

molecular coordinate system, the molecular orientation within the PVA film system must be established. This orientation can be determined from an analysis of the IR dichroic spectra. Figure 1 shows the IR absorption spectrum of FMNH- in an unstretched PVA film with a fit to a set of Gaussian functions using Origin 7.1. As shown in Figure 1, the bands at 1591, 1473, and 1391 cm-1 are most intense. However, the remainder of the spectrum appears relatively congested. The use of first and second derivatives of the IR spectrum (see Supporting Information, Figure S1) affords the determination of some subtle features in the spectrum. There are bands at 1672, 1623, 1593, 1573, 1537, 1500, 1472, 1439, 1413, 1394, and 1367 cm-1. The features at 1672, 1593, 1500, and 1391 cm-1 are in reasonable agreement with the one other indirectly measured IR spectrum reported by Birss et al.23 In that study, however, there is no band at 1472 cm-1. This may result from the use of D2O as a solvent, as observed by Zheng et al. in their resonance Raman study of reduced flavin in H2O and D2O solvents.18 A minimal deconvolution was achieved using the second derivative of the IR absorption spectrum (see Figure S1). In order to account for inhomogeneous broadening (i.e., primarily solvent broadening), a Gaussian line-shape function was used. The positions of the Gaussians were fixed and the width and amplitudes were allowed to float to achieve an initial set of Gaussian parameters. Then the peak positions were allowed to float to further minimize the residuals and achieve an optimal fit. Because only one IR spectrum and a few resonance Raman spectra of free reduced flavin have been reported, some difficulty exists in assigning the IR bands.18,23 For that matter, because Figure 1 is the first IR absorption spectra of FMNH- in a PVA film, there is no point of reference to identify any solvation effects caused by the PVA matrix. The closest comparison can be found in the LD study of oxidized flavins in PVA films.12 Matsouka and Norden measured the IR spectra of oxidized flavin in PVA films and in KBr.12 In comparison to the IR spectrum of oxidized flavin in KBr, the IR spectra in PVA films are less resolved and demonstrate some peak shifts and peak broadening. Quite possibly, similar solvent effects are present in the IR absorption spectra of FMNH- in a PVA film.

1394 1367 b

a Uncorrected raw computational results are in parentheses. Used in calculating the order parameters.

Assignment of IR Bands of FMNH- in PVA Films. In order to analyze the IR dichroic spectra and thus determine the orientation of the FMNH- in the PVA film, the assignment of the IR bands was required. This task presented a unique difficulty as a result of the paucity of reduced flavin IR spectra and the variation that exists in band assignment even in the current available literature on oxidized flavins.18-21 A brief review of the oxidized flavin literature and the one reduced flavin study suggested that the bands at 1672 and 1593 cm-1 can be assigned to normal modes with significant contributions from the C2 and C4 carbonyl stretching vibrations, respectively.18-21 In contrast to these bands, the assignment of the remaining bands varies from study to study. An additional complication in the IR band assignment arose from the possibility of hydrogenbonding interactions between the anionic reduced flavin and the PVA films. Any band predicted to involve contributions from N1, N3, N5, C2dO, and/or C4dO could possibly shift upon hydrogen-bonding with hydroxyl moieties in the PVA film.12 Such band shifts are seen in resonance Raman spectra of neutral and anionic reduced flavin.18 In a PVA film, such IR band shifts could result in a broadened and less resolved IR spectra. The normal mode calculations were performed using the DFT procedures (i.e., B3-LYP with a basis set of 6-31G*) on a reduced lumiflavin molecule with initial geometry optimized at the RHF/6-31G* level of theory. Because computationally determined IR frequencies are larger than experimentally observed IR frequencies, the calculated frequencies (in wavenumbers) were scaled by 0.93 to obtain good overlap of the computationally determined carbonyl stretch transition with the presumed experimentally determined carbonyl stretched transition. This allowed the calculated IR frequencies to be compared with the experimental IR spectra. Figure 1 shows the measured IR absorption spectrum of FMNH- in PVA films with a representative fit, the fitted set of Gaussian bands, and the computationally determined IR transitions. The results in Figure 1 are summarized in Table 1, which lists the experimental and the computational IR band frequencies in the 1750-1350 cm-1 range for FMNH-. Additionally, Table 1 contains the computationally determined assignments for each IR band. IR Dichroic Absorption Spectra and LDr Curve of FMNH- in PVA Films. Figure 2a shows the IR dichroic absorption spectra at parallel and perpendicular orientations relative to the polarization of the light. Figure 2b shows the IR reduced linear dichroic spectrum of FMNH- in PVA films.

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Figure 2. IR dichroic spectra and LDr curve of FMNH-. (a) IR dichroic spectra (A|, s and A⊥,0) of FMNH- in PVA films at Fs ) 20 ( 5 and (b) IR LDr curve of FMNH- in stretched PVA films (Nfilms ) 3) at Fs ) 20 ( 5.

LDr

As suggested by eq 7 above, the relatively constant IR values over the 1672, 1623, 1593, 1500, 1472, and 1394 cm-1 bands indicate that each of these IR transitions arise from differently polarized unique transitions. The pronounced change in the LDr value between 1473 cm-1 and 1394 cm-1 not only suggests the presence of at least one other unique transition between 1473 cm-1 and 1394 cm-1 but also could result from overlapping transitions. The derivative analysis of the IR spectrum (see Figure S1) and the computational determination of the normal modes of reduced flavin (see Figure 1) suggest the presence of a 1439 cm-1 transition. For that matter, the computational determination of the normal modes of reduced flavin demonstrates the presence of a few transitions between 1473 cm-1 and 1391 cm-1. Qualitatively, the small IR LDr value for the strong absorbance bands at 1593 cm-1 would indicate that this transition could have a large out-of-plane polarized component. However, if the strong 1593 cm-1 transition is assumed to lie parallel to the C4 carbonyl, then the small LDr value may arise from the C4 carbonyl lying out-of-plane. Furthermore, the large IR LDr value for the medium intensity absorbance band at 1439 cm-1, which consists of large contributions from ring-I modes and N5-H bending, indicates that this transition is dominated by an in-plane polarized component. If the isoalloxazine ring system is divided into two planes along the N5-N10 axis and the 1593 cm-1 transition is assumed to lie parallel to the C4 carbonyl, the small IR LDr value at 1593 cm-1 combined with the large IR LDr value at 1439 cm-1 would suggest that the FMNH- is not a planar molecule but perhaps a bent or “butterfly”-shaped molecule along the N5-N10 axis. This indirect result was also supported by the computational optimization of the reduced lumiflavin geometry prior to the normal mode calculations or the electronic transition dipole moment calculations. The optimized reduced lumiflavin was bent by 11-19° along the N5-N10 axis. Additionally, the “butterfly” conformation for reduced flavin is supported by previous computational studies and X-ray crystallography studies.18,24-26 Determination of the Orientation Axis and Parameters. The molecular orientation of the FMNH- in the PVA film must be established to determine the directions of the electronic transition dipole moments. In terms of the LD equations presented above, this requires the determination of the Szz, Syy, and the diagonal coordinate system (i.e., R0 for which Syz ) 0).

Figure 3. IR transition dipole moments for the corrected normal modes of reduced flavin at 1439, 1500, and 1593 cm-1

TABLE 2: Order Parametersa for Transition Dipole Moments in FMNHR0b

Szz

Syy

Sxx

66° ( 3 66° ( 1

0.372 ( 0.018 0.370 ( 0.013

-0.072 ( 0.021 -0.069 ( 0.001

-0.300 ( 0.009 -0.301 ( 0.012

a First row of calculations varies the transition angle (10°. The second row of calculations varies the LDr values from maximum to minimum with transition angle fixed. b Angle R0 at which the Syz ) 0.

These values and the orientation of the reduced flavin can be determined from the analysis of the IR dichroic spectra. Beyond the assignment of the IR bands to specific normal modes, the analysis of the IR dichroism requires the IR transition moment directions. These directions can be determined from the calculated normal modes of FMNH-. In order to solve eq 3, three transitions, the corresponding IR LDr, and the directions of those transitions need to be selected. The task of selecting the appropriate IR LDr values can be approached from a simple review of the features of the IR absorption spectra, the IR LDr spectrum, and from the transitions. Figure 3a-c presents the IR transition dipole moments for the calculated normal modes of reduced flavin at 1439, 1500, and 1593 cm-1. As presented above (see Figure 1 and Table 1), the 1593 cm-1 transition results primarily from the C4 carbonyl stretch. Thus, the vector sum of the component vectors of this transition should give a resultant vector with direction parallel to this stretch. The vector sum indicates that the direction of the IR transition dipole moment for the 1593 cm-1 band is rotated 15° clockwise relative to the C4 carbonyl. If the direction of the 1593 cm-1 transition is defined as 0° (i.e., z′ axis), the 1500 cm-1 transition direction is 131° counterclockwise to the z′ axis, and the 1439 cm-1 transition direction is 120° counterclockwise to the z′ axis. Computational studies have demonstrated that calculated IR transition dipole moment directions agree within 30° of experimentally determined directions.27,28 This information combined with the appropriate IR LDr values substituted in eq 3 will give Sy′y′, Sz′z′, and the nondiagonal coordinate system. By using the LDr values for the bands at 1593, 1500, and 1439 cm-1, the IR transition dipole moment directions for these bands, and eq 3, the order parameters Sy′y′, Sz′z′, and Sy′z′ were determined. From these order parameters in the nondiagonal system and eq 6, the diagonal xyz coordinate system and the diagonal order parameters Syy and Szz were determined. The IR LDr value for the 1593 cm-1 transition is 0.005 ( 0.002 for

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Figure 4. R0 in the molecular frame. (a) The z′ axis and the angle R0 defined in the isoalloxazine skeleton of the flavin. (b) An orientation triangle with the diagonal (Szz, Syy) values of FMNH-, RF, and MLF plotted.

reduced anionic flavin. The IR LDr value for 1500 cm-1 transition and the 1439 cm-1 transition are 0.021 ( 0.006 and 0.328 ( 0.371, respectively. Substitution of these values and the corresponding IR transition moment directions into eq 3 gives:

0.005 ( 0.002 ) 3[Sy′y′ sin2(0°) + Sz′z′ cos2(0°) + Sy′z′ sin (0°) cos(0°)] 0.021 ( 0.006 ) 3[Sy′y′ sin2(131°) + Sz′z′ cos2(131°) + Sy′z′ sin(131°) cos(131°)] 0.328 ( 0.371 ) 3[Sy′y′ sin2(120°) + Sz′z′ cos2(120°) + Sy′z′ sin(120°) cos(120°)] (8) As a result of the uncertainty in the calculated transition directions (i.e., 10-30°), we explored the sensitivity of the Sii parameters to the range of possible transition directions. The use of an in-house computer program also allows consideration of the physical limits of the Sii parameters and incorporation of eq 6 to determine the angle R0 and Syy and Szz (see Table 2). Table 2 shows the diagonal order parameters derived from the corresponding solutions to the above equations. The first row of results in Table 2 gives solutions of the above equations with LDr values fixed and variation in the calculated transition directions. The second row of results in Table 2 gives solutions of the above equations with the calculated transition directions fixed and variation in the LDr values. As a final check, the 1439 cm-1 transition was replaced by the 1463 cm-1, which has a different LDr and transition moment direction. The R0 and θ values obtained for the 290 and 350 nm transitions were the same to within experimental error as those for the transition triple containing the 1439 cm-1 transition (see Table S1, Supporting Information). The diagonal xyz axis system (i.e., the R0 value) and a corresponding set of diagonal order parameters Syy and Szz are shown in Figure 4. For comparison, the position of oxidized riboflavin (RF) and oxidized methyllumiflavin (MLF) are also shown in the orientation triangle (Figure 4b).12 The FMNHhas a rod-like orientation more similar to MLF than riboflavin. This can be understood as an effect of the repulsion between the negatively charged phosphate moiety at the end of the ribityl chain and the negatively charged pyrimidine ring of the isoalloxazine moiety. The repulsion may force the ribityl chain

Siddiqui et al.

Figure 5. Average UV/Vis LDr of FMNH- in PVA (Fs ) 25 ( 5), including error bars; isotropic normalized absorption spectra of FMNHin PVA (0; Fs ) 20 ( 5). The boxes and horizontal bars indicate the relevant transitions and associated LDr values.

to adopt a position relatively parallel to the long axis of the isoalloxazine moiety. UV/Vis Linear Dichroism of FMNH-. Figure 5 shows the reduced linear dichroism spectrum and isotropic absorption spectrum of FMNH- in stretched PVA. The LDr spectrum is the average of the spectra from 7 different samples with a Fs ) 25 ( 5. A comparison of the LDr spectrum and the isotropic spectrum indicates the presence of three unique transitions, one at 290 nm, one at 350 nm, and one at 440 nm. These absorption band positions are in reasonable agreement with literature values.29 For each absorption band, the LDr value is only a function of the angle θ between the transition and the molecular orientation axis (i.e., z axis). In other words, the LDr value indicates the relative alignment between the transition dipole moment and the z axis. Thus, a large LDr value would indicate that a particular transition is closely aligned with the z axis. Furthermore, across an isolated absorption band, the LDr value should be relatively constant. Qualitatively, the larger LDr values of the 350 and 440 nm transitions relative to the 290 nm transition indicate that they are relatively more aligned with the z axis. The solvent field of the PVA film induces a small shift of the absorption bands of the reduced FMNH- compared with those of the aqueous solution (see Figure S4, Supporting Information): the S0 f S1 band shifts from 420 nm in water/ glycerol to 440 nm in PVA, the S0 f S2 transition shifts from 344 to 350 nm, and the S0 f S3 transition shifts from 292 to 290 nm. With a dielectric constant for PVA of s ∼ 28, one would expect the flavin to exhibit absorption characteristics similar to an ethanol (s ∼ 25) or methanol (s ∼ 33) solution. In ethanol environment, the reduced flavin spectrum appears slightly red-shifted relative to the aqueous environment and shows less sensitivity to deprotonation at N1.29 The blue shift seen here may be a result of specific interactions with the PVA matrix that are absent in isotropic solution. A comparison of the averaged isotropic spectrum in the PVA film and the spectrum in aqueous solution shows some new features that arise from the PVA solvent. The isotropic absorption spectrum of FMNH- in PVA (see Figure 5) shows an additional small absorbance between 305 to 320 nm which is missing in the aqueous solution spectrum.30 The increased absorbance can be explained by differences in the thickness and anisotropy of the PVA itself (see Figure S2). While the absolute

Electronic Transition Dipole Moment Directions

J. Phys. Chem. B, Vol. 112, No. 1, 2008 125

TABLE 3: Experimentally Determined Transition Dipole Moment Directions in FMNHR0

λ (nm)

LDr

|θ|a

θb

66° ( 3

290 350 440

0.50 ( 0.03 0.78 ( 0.03 0.57 ( 0.03

42° ( 4 28° ( 4 39° ( 4

93° ( 4 (8° ( 4) 79° ( 4 (23° ( 4) 90° ( 4 (13° ( 4)

a Angles relative to the diagonal z axis. b Angles relative to the N5N10 axis toward the C4 carbonyl (i.e., CCW rotations). The solutions in parentheses do not compare reasonably with the LDr spectrum and/ or the theoretical calculations

TABLE 4: Calculated Electronic Transition Dipole Moment Directions of FMNH- in Various Solvents Using TDDFT at the B3LYP/6-31G* Level solvent ()

λcalc

θa

f

vacuum (1)

286 nm 375 nm 440 nm

50° 89° 75°

0.19 0.12 0.0015

a Angles relative to the N5-N10 axis toward the C4 carbonyl (i.e., CCW rotations).

absorbances between 305 and 320 nm are small, these absorbances are large enough to explain increased absorbance at ∼310 nm in the reduced flavin films. Qualitatively, the UV/vis LDr spectrum of FMNH- is positive over the measured wavelength region. This suggests that the transitions are primarily in-plane and polarized along the long axis of the molecule. Since the IR dichroism indicates that the molecule is not perfectly planar, the projection of the UV/vis transition dipole moments on the yz plane (i.e., in-plane) contributes more strongly to the LDr spectrum than those on the xz plane (i.e., out-of-plane). As a result, the out-of-plane projections of these transitions are relatively weak and are ignored in the analysis, as has been done in the case of oxidized and semiquinone flavins.12-14 In our case, inclusion of these projections would result in contributions that fall within the experimental error. Thus, eq 7 is sufficient for the determination of the electronic transition dipole moment directions.12-14 Determination of the Transition Dipole Moment Directions. By using the diagonal order parameters, the directions of the electronic transition dipole moments can be determined relative to the diagonal z axis. From the LDr spectra and the previous LD studies of oxidized and semiquinone flavin, the electronic transition dipole moments should lie along the long axis of the molecule. Since solution to eq 7 gives the angle θ for a transition relative to the diagonal z axis, there are two possible transition dipole moment directions per transition (see Table 3 and Figure S3). The unphysical pair can be eliminated by careful review of each result relative to the LDr spectrum and to theoretical calculations (see Table 4). Figure 6 depicts the calculated electronic transition dipole moment directions of reduced flavin in vacuum (dotted double arrow) and the experimentally determined (solid double arrow) electronic transition dipole moment directions of FMNH- in PVA films. The directions of the transitions from the LDr analysis are 90° ( 4° at 440 nm, 79° ( 4° at 350 nm, and 93° ( 4° at 290 nm with counterclockwise rotations with respect to the N5-N10 axis. As shown in Table 4, calculations at the B3-LYP/6-31G* level of theory support the assignment that the electronic transition dipole moments of the S0 f S1 and S0 f S2 transitions in FMNH- lie along the long axis of the molecule (i.e., Figure S3, case A) and are within 15° of the experimental results. These low-energy transitions appear to be very sensitive to the PVA solvent. The calculated wavelength for the S0 f S2 lies at 375

Figure 6. Electronic transition dipole moment directions in FMNH-: experimental (solid) and calculated with TDDFT using B3LYP/6-31G* (dotted).

nm compared with 350 nm for the film. This sensitivity to solvent conditions has been observed experimentally.29 The S0 f S1 band appears less sensitive to the PVA matrix, although the band is more difficult to analyze because of its broadness. On the contrary, the computed direction of the transition dipole for the low wavelength transition, S0 f S3, lies midway between the long and the short axes of the molecule, which is not in agreement with the LDr result. This discrepancy exists even when the TDDFT calculation includes 10 singly excited states (results not shown). The calculated short wavelength transition of FMNH- in vacuum is centered at 286 nm, which agrees well with the experimentally observed 290 nm transition in PVA. A few previous computational studies of the reduced anionic flavin are available. A ZINDO/s calculation suggested the electronic transition dipole of the low-energy transition at 359 nm lies along a line between C6 and N1, which is ∼135° counterclockwise with respect to the N5-N10 axis.31 This result would also suggest that the electronic transition dipole moment lies well off the long axis of the isoalloxazine moiety. Our results show that this is not the case. Recently, Prytkova et al.32 used TDDFT, TDHF, and INDO/S methods to estimate the excitedstate electronic structure of FADH-. They obtained very similar oscillator strengths as our calculations, but no estimates of the transition dipole moment directions were given. The S0 f S1 transition was considered too weak to be involved in the excitation of the cofactor, but no mention was made of the long wavelength tail of the flavin absorption beyond 400 nm. Our results show that the different computational approaches employed in our and other studies do not account for the LD results presented here. As such, a major result of this study is the resolution of the 350-450 nm absorption in FMNH- into two distinct transitions, as evidenced by their differing LDr values. All previous computational work has suggested that there might be a π f π* transition around 440 nm but that the oscillator strength for this transition was 100-fold less than the approximately 350 nm transition. Our TDDFT calculations also gave this result. If this were so, a solution of FMNH- should be colorless but is actually pale yellow to the eye, suggesting that the ab initio and semiempirical results are incorrect. Our LD results clearly support that a separate relatively bright transition is present in the visible region around 440 nm. This result may require the re-examination of previous photochemical and photobiological analyses that have made the one band assumption. For example, DNA photolyase is a flavoprotein that performs light-driven thymidine dimer repair following photoexcitation of a reduced anionic flavin.33 Several groups have probed the kinetics and underlying mechanism of

126 J. Phys. Chem. B, Vol. 112, No. 1, 2008 DNA photolyase by ultrafast spectroscopy using excitation wavelengths around 400 nm,10,34-37 and there appears to be no consensus on the interpretation of these data. None of the analyses cited take into account the near parallel nature of the 440 and 350 nm transition dipoles. This new information will certainly change the way we interpret the mechanism of lightdriven flavoproteins that utilized the reduced anionic cofactor. A comparison of the direction of the two lowest energy transition dipole moments with the transition dipole moments for the two lowest energy transitions for oxidized and semiquinone flavin reveals that all three oxidation states have nearly parallel transition dipole moment directions.14 This remarkable observation deserves comment. Eaton et al.14 obtained the transition dipole moments for oxidized and semiquinone FMN in flavodoxin crystals from LD studies on single crystals of flavodoxin. They attributed the coincidental overlap of the transition dipole moments for the different oxidation states to one electron reduction leading to the occupation of the lowest unoccupied π orbital of FMNox without any large change in the electronic structure of the molecule. We have measured the Stark spectroscopy of oxidized8,38 and semiquinone flavoproteins (Kodali, Siddiqui, and Stanley, in preparation). Interestingly, the change between ground and excited-state permanent dipole moments measured in the different oxidation states are very similar. We are presently obtaining Stark spectra for the reduced anionic flavin. From considerations of the transition dipole moment, we would expect that the change in permanent dipole moment for reduced anionic flavins will track the oxidized and semiquinone results. Further experiments are needed to determine if this is the case. Conclusions The UV/vis LD of partially oriented FMNH- in PVA films was measured over the 200-700 nm region and was resolved into contributions from three π f π* transitions. Assignments were based on an analysis of the IR LD spectrum that required the assignment of normal modes of FMNH- in the PVA film. These normal modes were assigned with guidance from TDDFT calculations. These calculations allowed computation of the normal modes and the IR transition dipole moment directions. From the UV/vis LD data, the electronic transition dipole moment directions have been experimentally determined: the directions of these transitions are 90° ( 4° at 440 nm, 79° ( 4° at 350 nm, and 93° ( 4° at 290 nm with counterclockwise rotations with respect to the N5-N10 axis. These directions are remarkably similar to those obtained in previous studies for the two lowest optically allowed transitions in oxidized and semiquinone flavin. The 440 nm band has been identified as a separate transition from the 350 nm absorption for the first time. Acknowledgment. We thank Mr. Elliott Echt of the Dupont Corporation for his beneficial suggestions. Dr. Todd Nelson of Merck helped to faciliate the transfer of the MACS anerobic chamber to our laboratory through Merck’s Institute for Science Education. We wish to thank Dr. Daniel Strongin for use of the FTIR spectrophotometer. We wish to thank Dr. Spiridoula Matsika for useful discussions and providing computer time through her UNIX accounts. We would like to thank the Petroleum Research Foundation (ACS-PRF 35353-G4) for partial support of this research. Supporting Information Available: Experimentally observed transition dipole moment directions, IR absorption spectra, UV/vis LDr, absorption spectra, possible electronic

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