Dynamics and Mechanisms of Ultrafast Fluorescence Quenching

We have interpreted the fluorescence quenching mechanisms of these .... Glucose oxidase (GOD) from Aspergillus niger was purchased from Nacalai Tesque...
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J. Phys. Chem. B 2000, 104, 10667-10677

10667

Dynamics and Mechanisms of Ultrafast Fluorescence Quenching Reactions of Flavin Chromophores in Protein Nanospace Noboru Mataga,* Haik Chosrowjan, and Yutaka Shibata Institute for Laser Technology, Utsubo-Hommachi 1-8-4, Nishi-ku, Osaka 550-0004, Japan

Fumio Tanaka Mie Prefectural College of Nursing, Yumegaoka, 1-1-1, Tsu 514-0116, Japan

Yasuzo Nishina and Kiyoshi Shiga Department of Physiology, Kumamoto UniVersity School of Medicine, Honjo, Kumamoto 860-0811, Japan ReceiVed: June 13, 2000; In Final Form: September 4, 2000

We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), D-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp.NH) or tyrosine (Tyr.OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90-100 fs) could be attributed to the compact stacked arrangement, Trp.NH.....F.....Tyr.OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck-Condon) f Fl (fluorescence) state conversion in PNS within the time resolution of the apparatus. The ultrafast FC f Fl conversion may be a coherent process coupled with intra-chromophore high-frequency modes leading to formation of vibrationally nonrelaxed or only partially relaxed Fl state, from which barrierless ET seems to occur. Fluorescence dynamics of DAOB have indicated faster initial decay in both blue and red sides of the spectrum contrary to other flavoproteins which showed practically wavelength-independent fluorescence dynamics. This result of DAOB is similar to those of photoactive yellow protein and visual rhodopsin although their reaction mechanism (twisting) is different from DAOB (ET). We have proposed a possible mechanism for this fluorescence dynamics of DAOB on the basis of an extremely compact stacked configuration of F...benzoate-...Tyr.OH which seems to undergo moderate frequency intermolecular vibration coupled with intra-chromophore high-frequency modes of F in the course of ET from Tyr.OH to excited F.

Introduction Flavoproteins with flavin chromophore (isoalloxazine) are enzymes ubiquitous in various biological systems where they undergo various important redox reactions.1 In most cases, those reactions of flavins in biological systems are not light driven. Nevertheless, studies on photoinduced reactions of flavins have been performed as models to facilitate the elucidations of the reaction mechanisms of those biological systems.1 On the other hand, although examples are rather few, some flavin enzymes seem to play also important roles in photobiological reactions. One of the well-known examples is the DNA photolyase which photorepairs a cyclobutane pyrimidine dimer produced by ultraviolet light in DNA. The DNA photolyase has the flavin chromophore in the reduced form, 1,5-dihydroflavin adenine dinucleotide (FADH2), and bind to damaged DNA and split the cyclobutane ring of the dimer. It has been directly proved by means of picosecond (ps) laser photolysis and timeresolved spectral measurements that splitting takes place with * Author to whom correspondence should be addressed.

very high efficiency owing to the electron transfer (ET) from the photoexcited FADH2 to the dimer.2 Another example is the blue light effect in the photosynthesis of plants. The CO2 absorption through the stoma of a plant leaf during the photosynthesis by red light seems to be regulated by flavoproteins excited with blue light,3 where flavoproteins are in the oxidized form and working as photosensor systems. In addition, there seem to be some other photobiological systems in which flavoproteins function as blue light photoreceptors, although direct experimental demonstrations of the relevant photoreactions by means of the time-resolved transient spectroscopy are scarce.4 When the flavin chromophore is in the oxidized form, it can act as a strong electron acceptor in the photoexcited state, in view of its molecular and electronic structures. Therefore, if such amino acid residues as tryptophan (Trp.NH) and tyrosine (Tyr.OH) are placed close to the flavin chromophore in protein nanospace (PNS), quenching of the flavin fluorescence due to the ET from the amino acid residue to the excited flavin chromophore can take place easily. Actually, the bright fluo-

10.1021/jp002145y CCC: $19.00 © 2000 American Chemical Society Published on Web 10/21/2000

10668 J. Phys. Chem. B, Vol. 104, No. 45, 2000 rescence of the isoalloxazine chromophore in solution is strongly quenched in PNS and there are many “nonfluorescent” or very weakly fluorescent flavoproteins. This is the most important characteristics of flavoproteins for their functions as photoreceptors. Furthermore, they are also very interesting and important model systems for elucidating the ultrafast reaction dynamics in PNS as in the case of the ultrafast primary photoreactions in the photosynthetic reaction centers and photosensory proteins studied extensively up to now. Experimental proof of the ET mechanism of the fluorescence quenching and elucidation of the ultrafast quenching dynamics in PNS of flavoproteins are rather scarce. We have previously tried to detect the photoinduced ET reaction by means of the picosecond (∼10 ps) time-resolved transient absorption spectral measurements on some flavoproteins and also solutions of flavin chromophores (lumiflavin (Lf), riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD)) with added quenchers such as indole and phenol corresponding to the amino acid residues Trp.NH and Tyr.OH, respectively.5,6 Actually, we have detected the transient absorption spectra which can be ascribed to the radical ion pair state formed by ET from a nearby Trp.NH to the excited flavin chromophore (F*) in PNS, i.e., F*.....Trp.NH f F-.....Trp.NH+, for flavodoxin, one of the typical “nonfluorescent” flavoproteins with FMN chromophore.6 We have observed also transient absorption spectra which might be due to neutral radical pair formed by photoinduced ET coupled with proton transfer (PT) in some chromophore solutions with added phenol (Ph.OH) and also in some flavoproteins,5,6 i.e., F*.....Ph.OH or F*.....Tyr.OH f F-.....Ph.OH+ or F-.....Tyr.OH+ f F.H.....Ph.O or F.H.....Tyr.O. However, clear-cut assignment of the product species in the latter case of the neutral pair formation was rather difficult because of their very broad and weak absorption spectra. Moreover, the direct observation of the reaction dynamics leading to the product formation was not possible in most cases because the main part of their photoinduced reactions took place in 100 fs-a few picosecond regimes. To examine directly the ultrafast ET reaction dynamics of the flavin chromophore in PNS of these “nonfluorescent” or very weakly fluorescent proteins, we have measured their fluorescence dynamics by means of the femtosecond up-conversion method and have published already a preliminary short report.7 On the other hand, we are examining also photoinduced primary processes of such photoactive or photosensory proteins as rhodopsin (Rh)8,9 and photoactive yellow protein (PYP)10,11 which play important roles in vision and negative phototaxis of the purple sulfur bacterium Ectothiorhodospira halophila, respectively, by means of the femtosecond fluorescence upconversion method. Both chromophores of these photobiologically important proteins undergo rapid photoisomerization in their primary reaction process although their molecular structures are quite different from each other. Both of their initial reaction from Franck-Condon (FC) excited-state toward twisted state (Tw) seem to be ultrafast coherent ones coupled with intrachromophore high-frequency modes as well as vibrational modes of environmental PNS.8-11 Although the reaction mechanism (ET) of the photoprimary process in the “nonfluorescent” flavoproteins is different from those of Rh and PYP (isomerization), both of them undergo the extremely fast photoinduced reaction in PNS and our previous investigations7 have indicated that not only Rh and PYP but also many “nonfluorescent” flavoproteins undergo such ultrafast barrierless and/or coherent process in PNS. Namely, the fluorescence Stokes shift of those flavoproteins seems to

Mataga et al. take place within the time resolution of the femtosecond upconversion apparatus (e100 fs)7 probably due to the coupling of the FC f Fl conversion with high-frequency vibrational modes of the chromophore and environmental protein and ET will proceed from such state competing with fluorescent transition. In the present article, we report detailed femtosecond fluorescence up-conversion studies on several “nonfluorescent” and very weakly fluorescent flavoproteins. We discuss the dynamics and mechanisms of the ultrafast fluorescence quenching reactions, taking into consideration the chromophore and surrounding amino acid residue configurations in PNS based on the available high-resolution X-ray crystallographic structures of flavoproteins. Experimental Section Riboflavin binding protein (RBP) was prepared from egg white as apoprotein and reconstituted by adding riboflavin according to the method by Rhodes et al.12 Excess riboflavin was carefully removed by repeated dialysis. Glucose oxidase (GOD) from Aspergillus niger was purchased from Nacalai Tesque (Kyoto, Japan) and purified according to Tsuge et al.13 Medium-chain Acyl-CoA dehydrogenase (MCAD) was purified from hog kidney as described by Gorelick et al.14 and Lau et al.15 The enzyme prepared was stored as a precipitate with ammonium sulfate in 0.1 M sodium phosphate at pH 7.6. The precipitate was dialyzed overnight in the same buffer before the fluorescence measurements. D-Amino acid oxidase-benzoate complex (DAOB) was purified from hog kidney according to the method reported.16 D-Amino acid oxidase (DAO) free from benzoate was prepared from the complex adding excess D-alanine.16 FMN and RF were purchased from Nacalai Tesque and purified by column chromatography with DEAE-cellulose.17 A buffer solution of flavoprotein sample was made to flow through a 1 mm cell, and a femtosecond up-conversion apparatus for the measurement of fluorescence dynamics was similar to that described elsewhere.7,18 The fwhm of the instrumental response was 210 fs. Measurements were made at room temperature (∼23 °C). In the case of DAOB, we examined temperature dependencies of the fluorescence decay curves near room temperature, for which we used the apparatus equipped with a homemade Peltier device. Observed fluorescence rise and decay curves were deconvoluted taking into consideration the instrumental response and reproduced by superposing exponential functions. Protein structures were obtained from Protein Data Bank (PDB), and redrawn with a software of Rasmol. Distances between atoms of isoalloxazine ring and nearby amino acid residues were calculated with the PDB coordinates. Results and Discussions We have tried hitherto to observe the femtosecond-topicosecond fluorescence dynamics of those “nonfluorescent” or very weakly fluorescent flavoproteins, RBP, GOD, MCAD, and DAOB, for which information on the high-resolution X-ray structures are available. Such information on the structures of PNS of the amino acid residues surrounding the flavin chromophore are very useful for the investigations on the quenching mechanisms of the chromophore fluorescence. In the following, we show detailed results of our studies on fluorescence dynamics of the above flavoproteins obtained by femtosecond fluorescence up-conversion method and discuss

Fluorescence Quenching Reactions of Flavin Chromophores

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10669

Figure 1. Structures of flavin chromophores: lumiflavin (Lf), riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD).

the dynamics and mechanisms of fluorescence quenching taking into consideration the structures of the chromophore and surrounding amino acid residues in PNS revealed by X-ray studies. Before that, we should give here a brief comment on some general features of stationary absorption and fluorescence spectra of flavin chromophores and flavoproteins. Characteristics of Absorption and Fluorescence Spectra of Flavin Chromophores and Flavoenzymes. Structures of flavin chromophores are indicated in Figure 1. Isoalloxazine (ISO) is responsible for the light absorption and emission of the flavin chromophores in the near-ultraviolet and visible regions. Ultrafast fluorescence quenching reactions of flavoproteins are caused by interactions between excited ISO with surrounding aromatic amino acid residues in PNS. Those flavin chromophores, Lf, RF, FMN, and FAD, in solutions show an absorption peak at 450-460 nm and a corresponding fluorescence band with peak at ca. 530 nm. Most flavoproteins show absorption peaks at the same wavelength as that of the free chromophore in solutions. Although the observation of the fluorescence band maxima of “nonfluorescent” flavoproteins is difficult, our results of fluorescence decay curve measurements at various wavelengths by femtosecond fluorescence up-conversion method as discussed later in this article indicate that the fluorescence wavelength ranges and peak positions of those flavoproteins are similar to those of the chromophore solutions. Namely, flavin chromophores such as FMN and FAD in aqueous solutions show fluorescence Stokes shift of ca. 3200 cm-1 and the above flavoproteins show also similar fluorescence Stokes shift. We have measured time profiles of fluorescence at various wavelengths covering fluorescence band of the flavin chromophores in aqueous solutions and detected indications of a faster decay at the short wavelengths and a little rise at the longest wavelengths owing to the time-dependent Stokes shift of the fluorescence caused by the solvation dynamics. However, as we show later, we could not recognize such an effect of the dynamic Stokes shift of fluorescence in flavoproteins examined here, even though their fluorescence Stokes shift is close to those of the chromophores in solutions. As discussed to some extent

Figure 2. Fluorescence dynamics of RBP excited at 410 nm and observed at various wavelengths and normalized at peak position. The instrumental response (fwhm ∼ 210 fs) is also indicated in the figure. The observed data are normalized and given by dots. Simulations of the observed decay curves with superposition of exponential functions are shown by solid lines.

in the Introduction and also will be discussed in more detail later, this result suggests an important problem on the nature of the flavoprotein fluorescence emitted in the course of the ultrafast quenching process being undergone in PNS. Dynamics and Mechanisms of Ultrafast Fluorescence Quenching Processes of Flavoproteins: RBP, MCAD, and GOD. In Figure 2, fluorescence dynamics of RBP excited at 410 nm and observed at various wavelengths from 497 to 634 nm and normalized at peak position are indicated together with the instrumental response. The rise and decay curves observed at various wavelengths were practically identical with each other and can be well reproduced by superposing three exponential functions, I(t) ) a1 exp(-t/τ1) + a2 exp(-t/τ2) + a3 exp(-t/ τ3). The contribution from the fastest component with decay time τ1 of about 100 fs was overwhelming (a1 g 90%). For example, at 605 nm: a1 ) 0.947, τ1 ) 93 fs, a2 ) 0.04, τ2 ) 1.02 ps, a3 ) 0.013, τ3 ) 10 ps (χ2 ) 0.15). This is an extremely short decay time compared with the decay time of several nanoseconds of the flavin chromophore in solutions. RBP is a globular monomeric protein of approximate dimensions 5 nm × 4 nm × 3.5 nm, and the RF chromophore is accommodated in a cleft (protein nanospace, PNS) of 2 nm wide and 1.5 nm deep, where the ISO ring of RF is stacked between Tyr.OH and Trp.NH placed in contact with the chromophore.19 In Table 1, the closest interatomic distances between ISO of RF and Tyr.OH as well as Trp.NH, and also interplanar distances between ISO ring and these aromatic amino acid residues taken from the X-ray structure analyses19 are indicated. The number-

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Mataga et al.

Figure 3. Numbering of atoms of amino acid residues and ligands in flavoproteins.

TABLE 1: Interatomic and Interplanar Distances between ISO of RF and Aromatic Amino Acid Residues in Close Proximity in the PNS of RBP19 amino acid atom no.

distance (Å)

chromophore

atom no.

ISO

(4) (3) (6) interplanar distance

Tyr-75

(7) (7) (7)

4.0 4.2 4.8 3.7

ISO

(9) interplanar distance

Trp-156

(6)

3.7 3.7

ings of the atoms of ISO in flavin chromophores, those of relevant amino acid residues and benzoate anion (in the case of DAOB) are shown in Figure 3. The Tyr.OH.....ISO.....Trp.NH stacked system of RBP is further surrounded by several Trp.NH residues.19 This arrangement of the chromophore and Tyr.OH as well as Trp.NH amino acid residues in PNS is supremely favorable for the ultrafast fluorescence quenching due to the ET and/or ET followed by PT from the amino acid residues placed close to the excited chromophore. In Figure 4, fluorescence dynamics of MCAD excited at 440 nm and observed at various wavelengths from 465 to 570 nm and normalized at peak position are indicated. Very similar time profiles of fluorescence were observed also when excited at 410 nm. All fluorescence dynamics observed at various wavelengths were practically the same and can be well reproduced by superposing three exponential functions, just as in the case of RBP. At 530 nm, for example; a1 ) 0.837, τ1 ) 299 fs, a2 ) 0.127, τ2 ) 1.09 ps, a3 ) 0.036, τ3 ) 42 ps (χ2 ) 0.12). We show also time profiles of fluorescence of GOD excited at 410 nm and observed at various wavelengths from 497 to 650 nm and normalized at peak position in Figure 5. Similar results were obtained also when excited at 430 nm. The fluorescence dynamics observed at various wavelengths were practically the same and can be well reproduced by superposing three exponential functions as in the case of RBP and MCAD. For example, at 620 nm: a1 ) 0.45, τ1 ) 413 fs, a2 ) 0.375, τ2 ) 1.85 ps, a3 ) 0.175, τ3 ) 6.02 ps (χ2 ) 0.06). Both fluorescence decays of MCAD and GOD are slower than that of RBP, but the fluorescence of MCAD decays faster than that of GOD. This results can be interpreted on the basis of the geometrical structures of flavin chromophore and surrounding aromatic amino acid residues in the PNS as discussed below.

Figure 4. Fluorescence dynamics of MCAD excited at 440 nm and observed at various wavelengths and normalized at peak position. The instrumental response (fwhm ∼ 210 fs) is also indicated in the figure. The observed data are normalized and given by dots. Simulations of the observed decay curves with superposition of exponential functions are shown by solid lines.

According to the X-ray crystallographic studies on MCAD20 and GOD,21 the ISO chromophore of FAD is surrounded also by several Tyr.OH and Trp.NH amino acid residues in PNS of both flavoproteins, as shown in Tables 2 and 3 and also in the three-dimensional figures for PNS of MCAD and GOD (Figure 6 A and B). However, these amino acid residues are placed neither so closely to ISO ring nor in stacked form sandwiching the ISO ring as in the case of RBP.19 Therefore, it is evident that the chromophore-amino acid residue interactions responsible for the photoinduced ET or ET coupled with PT are considerably weaker for MCAD and GOD compared with RBP, leading to the slower fluorescence decay dynamics. Furthermore, as shown in Figures 4 and 5 and as demonstrated above with the analysis of the fluorescence decay curves by superposition of the three exponential functions, the decay dynamics of MCAD is a little faster compared with that of GOD. The fastest component with decay time of ca. 300 fs is overwhelming (∼84%) and contributions from slow components of 1-10 ps regimes are very few (∼16%) in the decay curves of MCAD, while the contribution (∼45%) of the fastest component of ca. 400 fs is smaller than that (∼55%) of the components of 1-10 ps regimes in the case of GOD. It is not easy to interpret the above difference between MCAD and GOD regarding the fluorescence quenching dynamics. Nevertheless, we try here to point out some correlations between this difference and their PNS structures indicated in Tables 2 and 3. In both of the ISO-Tyr-375 pair of MCAD and ISO-Tyr515 pair of GOD, the phenyl ring part of ISO and Tyr.OH are

Fluorescence Quenching Reactions of Flavin Chromophores

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10671 TABLE 3: Interatomic Distances between ISO of FAD and Aromatic Amino Acid Residues in Close Proximity in the PNS of GOD21

Figure 5. Fluorescence dynamics of GOD excited at 410 nm and observed at various wavelengths and normalized at peak position. The instrumental response (fwhm ∼ 210 fs) is also indicated in the figure. The observed data are normalized and given by dots. Simulations of the observed decay curves with superposition of exponential functions are shown by solid lines.

TABLE 2: Interatomic Distances between ISO of FAD and Aromatic Amino Acid Residues in Close Proximity in the PNS of MCAD20 chromophore

atom no.

amino acid

atom no.

distance (Å)

ISO

(7) (8) (8) (9) (9)

Tyr-375

(6) (5) (6) (5) (6)

4.3 4.4 3.7 4.7 4.0

ISO

(3) (3) (3) (4)

Tyr-133

(1) (5) (6) (6)

5.3 4.6 4.2 4.2

ISO

(8) (8) (9) (9) (10) (11) (11)

Trp-166

(3) (5) (3) (5) (3) (2) (3)

5.4 5.8 5.1 5.1 4.3 4.4 3.9

relatively close to each other at similar intermolecular distances, leading to the similar ISO-Tyr.OH interactions responsible for the quenching of fluorescence due to the ET or ET coupled with PT. Also, the interactions in the ISO-Tyr-133 pair of MCAD and ISO-Tyr-68 pair of GOD do not seem to be much different from each other. In MCAD, the heterocyclic part of ISO with CdO group is close to the phenyl part of Tyr-133, and periphery phenyl part of ISO is close to hydroxy group of Tyr-68 in GOD. Thus, the ISO-Tyr.OH interactions in PNS of

chromophore

atom no.

amino acid

atom no.

distance (Å)

ISO

(8) (9) (9) (9) (10) (10)

Tyr-515

(6) (1) (6) (7) (1) (6)

4.2 4.2 3.7 6.9 4.3 4.3

ISO

(8) (8) (8) (9) (9)

Tyr-68

(3) (4) (7) (3) (7)

4.4 4.6 4.0 4.7 4.3

ISO

(3) (3)

Trp-111

(6) (7)

6.2 6.7

ISO

(6) (6) (10) (11) (11)

Trp-426

(7) (8) (3) (2) (3)

7.1 7.0 4.3 4.4 3.9

MCAD seem to be rather similar to those of GOD. However, concerning the ISO-Trp.NH interactions in the PNS, circumstances for MCAD for the ET quenching of fluorescence seem to be more favorable than for GOD. Namely, the ISO-Trp-111 pair of GOD is too distant to make sufficient interaction necessary for rapid photoinduced ET and also only a small part of the periphery phenyl ring of ISO is close to Trp-426 in GOD. Contrary to this, the pyrrole ring part of Trp-166 is placed more closely to the phenyl ring of ISO in MCAD as can be seen from Table 2. Since Trp.NH is much stronger electron donor compared to Tyr.OH,5 small difference of the geometrical arrangement in PNS can induce a considerable difference between MCAD and GOD regarding the fluorescence quenching dynamics by photoinduced ET. Thus, our results of the investigations on the ultrafast fluorescence dynamics of “nonfluorescent” or only very weakly fluorescent flavoproteins, RBP, MCAD, and GOD, are reasonably well correlated with their PNS structures revealed by X-ray analysis. Especially, the fluorescence quenching process of RBP is extremely fast reflecting the chromophore-amino acid residue arrangement in PNS where the ISO ring of RF is stacked between Tyr.OH and Trp.NH placed in contact with the chromophore19 as shown in Table 1. Ultrafast ET quenching of fluorescence due to the strong interactions between the chromophore and amino acid residues in the stacked structure can be barrierless and/or coherent process coupled with appropriate vibrational modes of the chromophore and environmental protein. Although it is rather difficult to observe the quantum beats in the ultrafast fluorescence decay dynamics under the present accuracy and timeresolution of the measurement, it is possible that the ultrafast decay was almost completed within first oscillation of the coupled vibrational mode in the case of RBP. As discussed already to some extent in the previous sections, before the fluorescence transition competing with the ultrafast ET quenching, the excited FC f Fl conversion within the time resolution of the apparatus should take place coupled with highfrequency vibrational modes of chromophore and environmental proteins, because we have not observed any effect of the dynamic Stokes shift in our measurements of fluorescence rise and decay dynamics at various wavelengths. The ultrafast FC f Fl conversion coupled with vibrational modes of the chromophore and environmental protein including coherent

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Mataga et al.

Figure 7. Ground-state absorption spectra of DAOB (A), DAO (B), and RBP (C).

Figure 6. Three-dimensional figure for the arrangements of the chromophore (ISO) and surrounding aromatic amino acid residues in the PNS revealed by X-ray crystallographic studies: (A) MCAD,20 (B) GOD,21 (C) DAOB.22,23 These figures indicate just images of threedimensional arrangements of chromophore and amino acid residues in each protein. Quantitative comparisons of intermolecular distances among these proteins are made in the text using the distances given in the Tables 2, 3, and 5.

process can give vibrationally unrelaxed or only incompletely relaxed Fl state. Such Fl state formation may be followed immediately by ultrafast ET quenching process competing with fluorescence radiative transition as described above. It should be noted here that, despite the strong interaction of the chromophore with aromatic amino acid residues in stacked form where their molecular planes are almost in contact, we cannot recognize any extra absorption band due to charge transfer (CT) interaction in the ground state for RBP, as shown

in Figure 7. Nevertheless, the photoexcitation of RBP leads to the ultrafast ET or ET-coupled-with-PT reactions. Another important issue in relation to the photoexcitation of RBP as well as other flavoproteins are their rather efficient photocycle proceeding in their PNS. Namely, in this “nonfluorescent” or very weakly fluorescent flavoproteins, the radical pairs formed by ultrafast ET or ET coupled with PT seem to return to the original ground state by efficient back reactions in the PNS, because these flavoproteins are very stable for rather long irradiation by laser pulse in the fluorescence up-conversion measurements. Contrary to this, when the flavin chromophores in solution are irradiated under the presence of high concentration quenchers such as Trp.NH or indole as well as Tyr.OH or phenol, they easily undergo the photodecomposition caused by ET or ET coupled with PT. The stable photocycle under repeated irradiation is the most important characteristics of photoresponsive protein working in such photobiological reactions as photosynthesis and various photosensory actions. As discussed in the Introduction, there are some flavoproteins actually working in photobiological reactions, showing such characteristics. However, the flavoproteins we have studied here are not such ones working in photobiological reactions. Nevertheless, they show rather similar characteristics as discussed above. This result means that such important characteristics of the photoresponsive protein seems to be determined by the ingenious functions of the PNS which can work not only in dark reactions but also in photoinduced reactions. In the case of MCAD and GOD, their fluorescence decay dynamics are not so fast as in the case of RBP, reflecting their chromophore-aromatic amino acid residue arrangements in PNS where the amino acid residues are placed neither so close to ISO chromophore of FAD nor in stacked form as in the case of RBP, leading to the weaker chromophore-amino acid residue interactions compared with RBP. Nevertheless, the overwhelming contribution (ca. 84%) of the fastest component with decay time τ1 of ca. 300 fs in the analysis of the fluorescence decay dynamics by superposing three exponential functions for MCAD suggests that the ET quenching reaction here is also barrierless. Although the fluorescence decay dynamics of GOD is a little slower compared with that of MCAD, the observed result (a1 ∼ 45%, τ1 ∼ 410

Fluorescence Quenching Reactions of Flavin Chromophores

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10673 TABLE 4: Fluorescence Dynamics of DAOB Observed at Various Wavelengths and Reproduced by Superposing Two Exponential Functions, I(t) ) a1 exp(-t/τ1) + a2 exp(-t/τ2) wavelength (nm)

a1

a2

τ1 (fs)

τ2 (ps)

χ2

480 485 490 510 530 550 580 600 630 640

0.815 0.71 0.60 0.22 0.337 0.36 0.26 0.25 0.486 0.47

0.185 0.29 0.40 0.78 0.663 0.64 0.74 0.75 0.514 0.53

300 420 506 942 1486 1460 940 877 840 713

1.9 4.23 4.46 4.47 4.95 5.0 4.52 4.40 6.46 7.34

0.74 0.62 0.64 0.10 0.04 0.05 0.06 0.17 0.35 1.05

TABLE 5: Interatomic Distances between ISO of FAD and Aromatic Amino Acid Residues in Close Proximity in the PNS of DAOB22,23 chromophore

Figure 8. Fluorescence dynamics of DAOB excited at 430 nm and observed at various wavelengths and normalized at peak position. The instrumental response (fwhm ∼ 210 fs) is also indicated in the figure. The observed data are normalized and given by dots. Simulations of the observed decay curves with superposition of exponential functions are shown by solid lines.

fs) indicates that the ET quenching reaction is still very fast and probably barrierless. Further, since we could not observe any indication of the dynamic Stokes shift in the fluorescence rise and decay dynamics observed at various wavelengths also for MCAD and GOD as shown in Figures 4 and 5, respectively, the ultrafast excited FC f Fl conversion seems to take place within the time resolution of the apparatus also for these flavoproteins. Accordingly, as discussed above for the case of RBP, it seems to be possible that the ultrafast FC f Fl conversion leads to the formation of the vibrationally unrelaxed or only incompletely relaxed Fl state followed by ET quenching competing with fluorescence transition. However, in view of the longer fluorescence decay times of these flavoproteins compared with RBP, such possibility of the ET reaction from vibrationally unrelaxed state may be a little smaller. Dynamics and Mechanisms of Ultrafast Fluorescence Quenching Processes of Flavoproteins: DAOB. In Figure 8, fluorescence dynamics of DAOB excited at 430 nm and observed at various wavelengths from 480 to 640 nm and normalized at peak position are indicated together with the instrumental response. For this flavoprotein, the fluorescence dynamics at various wavelengths can be well reproduced by superposing two exponential functions, I(t) ) a1 exp(-t/τ1) + a2 exp(-t/τ2). Further, the fluorescence decay dynamics of DAOB shows a systematic wavelength dependence as can be seen from Figure 8 and Table 4. That is, the fluorescence dynamics of DAOB

atom atom atom distance no. amino acid no. benzoate- no. (Å)

ISO

(4) (4) (5) (7) (13)

ISO

(5) (5) (7) (7) (8) (15) (16)

ISO

(7) (8) (9) (9) (15)

Tyr-224

(3) (7) (3) (3) (3)

Tyr-224

(2) (2) (3) (3) (7) (7)

Tyr-228

(4) (4) (4) (7) (7)

Tyr-228

(2) (2) (3) (3) (7)

6.8 6.6 6.6 6.5 6.5 benzoate-

(5) (6) (7) (9) (9) (9) (9)

3.5 3.3 3.2 3.2 3.5 3.0 3.3

benzoate-

(7) (8) (1) (6) (5) (6)

3.5 3.6 3.5 3.3 3.7 3.3 5.7 5.2 6.0 4.6 4.8

benzoate-

(2) (3) (2) (8) (8)

4.2 4.4 3.5 3.7 3.0

show faster initial decay in both blue and red sides of the spectrum, contrary to those of other flavoproteins discussed in the preceding section, RBP, MCAD, and GOD, where the fluorescence decay dynamics observed at various wavelengths was practically identical. To elucidate the mechanisms underlying this peculiar fluorescence dynamics of DAOB, we have examined the relation between the fluorescence dynamics and the arrangements of its chromophore and surrounding amino acid residues in PNS revealed by X-ray structural analysis.22,23 The interatomic distances between ISO of FAD and aromatic amino acid residues as well as benzoate in close proximity are shown in Table 5 and their three-dimensional arrangements in the PNS are indicated in Figure 6C. A very special feature in this three-dimensional structure is the closely stacked configuration of ISO.....benzoate-.....Tyr-224. We can see from Table 5 that the interplanar distances between ISO and benzoate are quite short (3.0-3.5 Å). Especially, the negatively charged (deprotonated) carboxyl group of benzoate is in contact with the central ring of ISO at very short distance of 3.0-3.2 Å. Such an extraordinary close contact between aromatic com-

10674 J. Phys. Chem. B, Vol. 104, No. 45, 2000 pounds such as ISO and benzoate may be impossible in solutions without any such special interaction as the strong charge-transfer complex formation. However, we cannot recognize such indication of the strong charge-transfer complex formation in the absorption spectra of DAOB in Figure 7. Absorption spectra of DAOB are a little red-shifted and broad vibrational structure in the longest wavelength absorption band becomes somewhat conspicuous compared with those of DAO and RBP, but spectral change attributable to such strong charge transfer complex formation cannot be observed in Figure 7. This result is quite reasonable because both ISO and benzoate seem to function as electron acceptors. The spectral change of DAOB indicated in Figure 7 may be caused by slight shifts of the energy levels of the ground and excited electronic states due to the “forced” very close contact of ISO with benzoate. Such an extraordinary close contact impossible in solution might be realized in this protein, presumably to attain minimum change in the free energy of the entire protein system. It should be noted here that, although RBP has a stacked structure where ISO is sandwiched by Trp.NH and Tyr.OH, the interplanar distance is a little longer (3.7 Å). At any rate, in the stacked configuration of ISO‚‚‚‚‚benzoate-‚‚‚‚‚Tyr-224 where Tyr-224 is placed fairly close to benzoate (3.3-3.7 Å), fluorescence quenching process due to ET from Tyr-224 to excited ISO may be facilitated by the presence of the conjugate π-electron system of the intervening benzoate presumably due to a kind of superexchange mechanism via benzoate. Since Tyr-228 is also rather closely placed to bonzoate, it may contribute also to some extent to the ET quenching of the fluorescence. In relation to such mechanism of fluorescence quenching reaction, it should be noted here that the fluorescence decay dynamics of DAO free from benzoate was reported to be single exponential with a lifetime of 44 ps.24 We have examined also DAO by means of the fluorescence up-conversion method in this work and observed essentially the same single-exponential decay with a lifetime of 40.5 ps. This decay time is much longer compared with those of DAOB shown in Figure 8 and Table 4, and does not contradict the above suggested mechanism of the enhancement of the ET rate by the intervening benzoate. As stated in the Introduction and in previous sections on RBP, MCAD, and GOD, we could not observe any effect of the dynamic Stokes shift in our measurements of fluorescence rise and decay dynamics at various wavelengths. That is, the fluorescence rise dynamics observed at various wavelengths from blue edge to red edge of the fluorescence spectrum was practically the same, which means that the excited FC f Fl conversion coupled with intra-chromophore vibrations and those of the environmental protein takes place within the time resolution of our measurements. The Fl state produced by such ultrafast conversion seems to undergo rapid quenching caused by ET or ET coupled with PT from the nearby aromatic amino acid residues to the excited flavin chromophore. Contrary to the fluorescence dynamics of the other flavoproteins discussed above, DAOB shows wavelength dependence of the fluorescence decay dynamics, as shown in Figure 8 and Table 4. Namely, faster initial decay of the fluorescence was observed in both blue and red sides of the spectrum. This wavelength dependence of the fluorescence dynamics is not due to the usual dynamic Stokes shift but is similar to those observed in the fluorescence decay dynamics of the wild-type PYP (photoactive yellow protein)10,11 and bovine Rh (rhodopsin).8,9 The faster initial decay of the fluorescence in both blue and red sides of the spectrum in the case of PYP and Rh has been

Mataga et al. interpreted on the basis of the coupling of the intrachromophore high-frequency vibrational modes (presumably some stretching mode) with the twisting of the chromophore leading to the isomerization.8-11 Namely, the faster initial decay of the fluorescence in blue and red sides of the spectrum may be due to the slight narrowing of the fluorescence band shape as the displacements of the high-frequency modes coupled with the twisting initially decrease slightly along the reaction coordinate of the isomerization.9,11 The fluorescence quenching mechanism of flavoproteins is not the geometrical structural change by twisting but ET or ET coupled with PT from aromatic amino acid residue to the excited chromophore molecule. Even in the latter case of the ET coupled with PT, fluorescence quenching presumably takes place in the ET process and PT occurs from cation radical to anion radical in the ion radical pair in some cases.25 However, as discussed in the next section, there is another case of the ET coupled with PS (proton shift) or PT in the excited state of hydrogen bonding systems, (D*-H...A) or (D-H...A*). Namely, a slight shift of proton from D-H to A in the excited hydrogen-bonded system facilitates greatly the ET state formation, or the ET state formation by a slight proton shift induces a large-scale proton shift or PT. In these cases, ET and PS or PT are more intimately coupled with each other.25,28-30 In any case, such a slight narrowing of the fluorescence band shape due to the decrease of the displacements of the high-frequency modes of the chromophore coupled with a little slower motion like twisting modes along the reaction coordinate in the early stage of the reaction immediately after FC f Fl conversion may not be expected in those flavoproteins, RBP, MCAD, and GOD. In view of the rather complex structure of DAOB with the ISO.....benzoate-.....Tyr-224 stacked configurations formed by added benzoate to the protein, a slight inhomogeneities in the distribution of PNS structures may be possible. In one kind of PNS the system may undergo mainly barrierless fast reactions in several hundreds femtosecond regimes but in another kind of PNS the system can undergo only slower reactions in several picosecond regimes crossing barriers. To examine such possibilities, we have measured temperature dependencies of the fluorescence decay curves of DAOB. However, time profiles of the fluorescence dynamics in hundreds femtosecond-10 ps regime showed exactly no temperature dependence both at the top region and bottom region of the fluorescence spectrum when temperature was changed from 20 to 5 °C. This result indicates that the nonexponential decay dynamics is due to the intrinsic barrierless reaction.26 Although the exact mechanism underlying the peculiar wavelength dependence of the fluorescence decay curve of DAOB is not very clear at the present stage of the investigation, we suggest here a possible correlation between the special PNS structure of DAOB and the peculiar wavelength dependence of the fluorescence decay dynamics. Namely, the negatively charged (deprotonated) carboxyl group of benzoate is in extraordinary close contact with the central ring of ISO at the distance of 3.0-3.2 Å and Tyr-224 is also placed very close to the benzoate at the distance of 3.3-3.7 Å in the stacked configuration, ISO.....benzoate-.....Tyr-224. In the course of ET from Tyr-224 to the excited ISO, the ISO negatively charged by ET will undergo strong repulsive interaction with the negatively charged carboxyl group of benzoate very closely placed to ISO. This strong repulsion will induce oscillations in the stacked molecular configuration in the course of the photoinduced ET process. That is, this oscillation in the stacked

Fluorescence Quenching Reactions of Flavin Chromophores molecular configuration will play an important role in the ET process along the reaction coordinate. The intra-chromophore high-frequency modes will facilitate the ultrafast FC f Fl conversion also in the case of DAOB. Immediately after the FC f Fl conversion, the chromophore high-frequency modes will be coupled with the oscillation in the stacked molecular configuration through the fluctuation of charge distribution in the ISO chromophore induced by the oscillation of the negatively charged benzoate in the course of the ET process. These vibrations in the chromophore and stacked configurations may interact further with oscillations of the environmental protein in the PNS. In this way, in the early stage of the fluorescence decay dynamics after the FC f Fl conversion, decrease of the displacements of the chromophore hig-frequency modes coupled with oscillations in the stacked configuration along the reaction coordinate of the ET process will induce a little faster decay in the early stage in both blue and red sides of the fluorescence spectrum. It should be an interesting problem to examine this interpretation by modifying the structure of the benzoate by substitution, etc. On the Mechanisms of ET and ET Coupled with PT between Excited Flavin Chromophore and Amino Acid Residues in PNS. Mechanisms and dynamics of excited-state ET reactions25,27 and ET-coupled-with-PT reactions25,27 in solution have been investigated extensively by means of nanosecond, picosecond, and femtosecond laser spectroscopic studies as well as theoretical inquiries. Owing to these studies, many important fundamental mechanisms have been elucidated although there still remains some elusive problems which should be made clear.25,27 Fluorescence quenching reactions of such flavin chromophore as Lf and RF by indole or phenol in various solutions were studied previously,5 results of which showed that the reactions in chloroform and ethanol solutions were very rapid (diffusioncontrolled). The driving force for the quenching reaction (free energy gap (-∆GCS) for the charge separation (CS) in the excited state of the flavin chromophore (F)) was estimated to be ca. 0.7 eV and ca. 1 eV for the F*.....indole pair in chloroform and ethanol solutions, respectively, assuming the encounter distance to be ca. 7 Å.5 For the F*.....phenol pair, because of the higher oxidation potential of phenol, this energy gap may be fairly smaller than for the F*.....indole pair. However, for the F*.....amino acid residue (Trp.NH and Tyr.OH) pairs placed in close proximity in PNS, as indicated in Tables 1-3 and 5, the F*.....quencher distances are much shorter than 7 Å, resulting in larger energy gaps for CS due to the larger coulomb stabilization in the ion-pair state and the stronger intermolecular overlap interaction (larger tunneling matrix element) responsible for the electron transfer. Therefore, not only for the F*.....Trp.NH pair but also for the F*.....Tyr.OH pair, barrierless ultrafast ET reaction can be realized in PNS as actually observed for the flavoproteins examined in this work. Moreover, when there exists hydrogen-bonding interaction between ISO of F and Trp.NH or Tyr.OH, the ET from the proton donor amino acid residue to the hydrogen-bonded ISO in the excited state can be facilitated by the hydrogen-bonding interaction. Namely, we showed for the first time that the intermolecular hydrogen-bonding interaction frequently induces fluorescence quenching especially when two conjugate π-electronic systems were directly combined by hydrogen-bonding interaction in nonpolar solvents.28 We proposed the CT interaction between the proton donor (D-H) and acceptor (A) in the

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10675 excited state of the hydrogen-bonding complex, leading to the formation of nonfluorescent ET state (D+-H.....A-), as a possible mechanism of the quenching28 and proved it by means of picosecond-femtosecond time-resolved transient spectroscopic measurements on several typical hydrogen-bonding systems.25,29 For example, in the case of 1-aminopyrene (D-H)-pyridine (A) system,29c a slight shift of proton from D-H to A in the hydrogen-bonded system in the excited state (D*-H.....A) seems to facilitate greatly the formation of ET state (D+-H.....A-) through which the excited state is deactivated. On the other hand, in the case of the 1-pyrenol (D-H)-pyridine (A) hydrogenbonded system in nonpolar solvent,29d a slight proton shift (PS) from D-H to A in the excited state seems to induce the ET state formation, followed by a large-scale proton shift or proton transfer (PT) state formation leading to the deactivation to the ground state. Furthermore, the ET and PS or ET and PT coupled processes in the excited state can take place not only in the typical hydrogen-bonding systems as described above but also takes place in the case of the aromatic hydrocarbon (A)-secondary aromatic amine (D-H) exciplexes in nonpolar solvent, where a special hydrogen-bonding interaction seems to exist between the π-electron system of the aromatic hydrocarbon and the >N-H of the secondary aromatic amine in the pair, (A-.....HD+) f (A-H.....D).25,27b,30 Thus, even in the case of the direct ET interactions between ISO and aromatic amino acid residues in flavoproteins (RBP, MCAD, GOD), except for DAOB where a more complex mechanism of photoinduced ET via intervening benzoate ion seems to prevail, there are various possible mechanisms of ET and ET coupled with PS or PT reactions between excited flavin chromophore and amino acid residues, especially when the reactants pairs are placed in close proximity in the PNS of those flavoproteins. It does not seem to be easy to quantitatively identify the relevant mechanism of the photoinduced reactions for each flavoprotein examined here. For this purpose, in addition to the femtosecond-picosecond fluorescence dynamics studies, femtosecond-picosecond transient absorption studies are of crucial importance. However, at the present stage of the investigation, we have no reliable results of time-resolved transient absorption spectral studies in femtosecond-picosecond regime related to the present fluorescence dynamics studies on flavoproteins. Nevertheless, we should give here brief discussions on our previous transient absorption spectral studies of some flavoproteins with 10 ps laser photolysis method,5,6 where we compared transient absorption spectra of Lf or RF solutions added with electron-donating quenchers, indole and phenol, with those of some flavoproteins. The transient absorption spectrum of flavodoxin (FD), with stacked structure of PNS similar to that of RBP where ISO of FAD is sandwiched by Trp.NH and Tyr.OH, immediately after excitation was very similar to that of the methanol solution containing 0.15 mM of RF and 0.23 M of indole. Namely, the transient absorption spectra could be interpreted as a superposition of the absorption bands of anion radical of flavin chromophore and cation radical of Trp.NH.6 We examined also the transient absorption spectrum of RBP by means of the same laser photolysis method.6 The observed spectrum immediately after excitation was very broad. It was rather similar to the transient absorption spectrum of ethanol solution containing 28 µM of Lf and 0.4 M of phenol but the band in the shorter wavelength region seems to resemble somewhat to that of FD. Presumably, ultrafast ET coupled with PT from Tyr.OH to excited ISO seems to take place mainly but

10676 J. Phys. Chem. B, Vol. 104, No. 45, 2000

Mataga et al.

TABLE 6: Interatomic Distances between ISO of FAD and Aromatic Amino Acid Residues in Close Proximity in the PNS of FD31 chromophore

atom no.

amino acid

atom no.

distance (Å)

ISO

(7) (7) (7) (9) (10) (11) (12) (15) (16)

Tyr-98

(1) (5) (7) (7) (7) (7) (5) (5) (5)

3.7 3.8 4.8 3.6 3.5 3.6 3.4 3.5 3.3

ISO

(8) (9) (10) (10) (11) (11)

Trp- 60

(5) (5) (5) (6) (5) (6)

4.4 3.9 3.8 3.7 4.1 4.0

the ET from Trp.NH to the excited ISO might occur also to some extent, in competition with the main process. We can see from Table 1 that, because the -OH group of Tyr-75 is very closely placed to CdO groups as well as >N-H group of ISO, the ultrafast ET coupled with PT can take place easily but also, ultrafast ET from Trp.NH can compete with it to some extent depending upon the fluctuations in the stacked structure. According to the X-ray structural analysis on FD,31 the closest distance between the -OH group of Tyr.OH and the heteroatom (-N)) of ISO, as indicated in Table 6, is rather large, so that the ultrafast ET coupled with PT does not seem to be feasible. However, owing to the interplanar close contact between Trp.NH and ISO, ultrafast ET can take place easily. On the other hand, according to our previous transient absorption spectral measurements on GOD and DAOB by means of the 10 ps laser photolysis method,6 the transient absorbance was too weak to observe the spectra.6 The intermediate states produced by photoinduced ET or ET coupled with PS as well as PT may be too short-lived to observe the spectra due to the ultrafast deactivation to the ground state by back reaction. In any case, for the identification of the reaction intermediates and elucidation of their dynamic behaviors, detailed transient absorption spectral measurements in femtosecond-picosecond regimes are necessary. Concluding Remarks We have studied excited-state dynamics of several “nonfluorescent” or only very weakly fluorescent flavoproteins by means of femtosecond fluorescence spectroscopy for the first time, and have revealed the following results concerning their ultrafast fluorescence dynamics and underlying reaction mechanisms in the PNS (protein nanospace). (a) Although the flavoproteins are rather ubiquitous in various biological systems, those examined here are not working in photobiological reactions. Nevertheless, they undergo ultrafast and very efficient fluorescence quenching reactions probably due to ET and ET coupled with PS or PT to excited flavin chromophore (F; Lf, RF, FMN, and FAD with ISO (isoalloxazine ring)) from aromatic amino acid residues closely placed in PNS when F is excited by light absorption. (b) The proposed fluorescence quenching mechanisms due to ET and ET coupled with PS or PT between F* and Trp.NH or Tyr.OH in PNS are supported by the available X-ray structures of those flavoproteins. Namely, for the more closely placed F.....Trp.NH or Tyr.OH pair with the more favorable

mutual orientation, the faster fluorescence decay dynamics due to the ET reaction has been observed. (c) The wavelength of the S1 r S0 absorption band peak of the chromophore F in aqueous solution is approximately the same as that of the flavoproteins and, moreover, the Stokes shift of the chromophore fluorescence in aqueous solution is approximately the same as that of flavoprotein. We have observed the typical effect of the time-dependent Stokes shift of fluorescence due to solvation dynamics in aqueous solution of F by femtosecond fluorescence up-conversion method. However, we could not recognize such an effect in the flavoproteins, i.e., the fluorescence dynamics observed at various wavelengths were practically the same. This means that the excited FC f Fl state conversion takes place coupled with high-frequency modes of the chromophore and environmental protein within the time resolution of the apparatus. Such ultrafast FC f Fl conversion probably including coherent process may give vibrationally unrelaxed or only incompletely relaxed Fl state, from which an ultrafast ET quenching process seems to take place. (d) The above-described photoinduced primary processes of the flavoproteins which are not working in such photobiological reactions as those of the bacterial photosynthetic reaction centers,32 photoresponsive or photosensory rhodopsin8,9 and PYP,10,11 etc., are similar to those of the typical photobiological proteins. This seems to indicate that, in such behaviors of the chromophore-protein complexes, the protein environment in the PNS plays predominantly important roles. (e) Among the flavoproteins examined here, DAOB shows a peculiar fluorescence dynamics different from the others. Namely, DAOB shows faster initial decay of fluorescence in both blue and red sides of the spectrum, while fluorescence dynamics observed at various wavelengths were practically the same in the case of the other flavoproteins examined here. In DAOB, the negatively charged benzoate- is in an extraordinary close contact with the central ring of ISO at the distance of 3.0-3.2 Å and Tyr.OH is also placed very close to benzoate- at the distance of 3.3-3.7 Å in the stacked configuration. The ISO negatively charged by ET will undergo strong repulsive interaction with the negatively charged benzoate- very closely placed to ISO-. This strong repulsion may induce oscillations in the stacked molecular configuration which will couple with the intra-chromophore high-frequency modes of ISO immediately after FC f Fl conversion, through the fluctuation of charge distribution in ISO induced by the oscillation of the negatively charged benzoate- in the course of the ET process. Thus, in the early stage of the fluorescence decay dynamics after the FC f Fl conversion, a slight decrease of the displacements in the chromophore high-frequency modes coupled with oscillations in the stacked configuration along the reaction coordinate of ET process will induce a little faster decay in the early stage in both blue and red sides of the spectrum. This result is very analogous to those of the wavelength dependence of the fluorescence decay dynamics in the early stage in the case of PYP10,11 and Rh.8,9 (f) To go beyond the descriptive stage of the present investigations on flavoproteins, it is necessary to establish a quantitative kinetic model for the fundamental mechanisms of fluorescence quenching reactions in PNS. However, these protein systems are not homogeneous fluid or solid solutions, and environmental amino acid residues in protein will play more specific roles than the bulk solvent. To take into account such effects of protein environment on the photoinduced ET reaction, we need molecular dynamics simulations. Moreover, there exist various cases of strong donor-acceptor interactions responsible

Fluorescence Quenching Reactions of Flavin Chromophores for the ultrafast ET between the excited chromophore and aromatic amino acid residues as described in this article, to which conventional or standard ET theories will not be applicable. These are very important fundamental problems which we are planning to examine in our forthcoming project. Acknowledgment. The authors express their thanks to the reviewers for their helpful discussions. References and Notes (1) For example: FlaVins and FlaVoproteins; Yagi, K., Yamano, K., Eds.; Japan Scientific Societies Press: Tokyo and University Park Press: Baltimore, MD, 1980. (2) (a) Okamura, T.; Sancar, A.; Heelis, P. F.; Begley, T. P.; Hirata, Y.; Mataga, N. J. Am. Chem. Soc. 1991, 113, 3143. (b) Kim, S.-T.; Heelis, P. F.; Okamura, T.; Hirata, Y.; Mataga, N.; Sancar, A. Biochemistry 1991, 30, 11262. (3) (a) Hsiao, T. C.; Allaway, W. G.; Evans, L. T. Plant Physiol. 1973, 51, 82. (b) Ogawa, T.; Ishikawa, H.; Shimada, K.; Shibata, K. Planta 1978, 142, 61. (4) (a) Song, P. S.; Fugate, R. D.; Brigs, W. R. In FlaVins and FlaVoproteins; Yagi, K., Yamano, K., Eds.; Japan Scientific Societies Press: Tokyo and University Park Press: Baltimore, MD, 1980; p 443. (b) Song, P. S. In Blue Light Syndrome; Senger, H., Ed.; Springer-Verlag: Berlin, 1980; p 157. (5) Karen, A.; Ikeda, N.; Mataga, N.; Tanaka, F. Photochem. Photobiol. 1983, 37, 495. (6) Karen, A.; Sawada, M. T.; Tanaka, F.; Mataga, N. Photochem. Photobiol. 1987, 45, 49. (7) Mataga, N.; Chosrowjan, H.; Shibata, Y.; Tanaka, F. J. Phys. Chem. B 1998, 102, 7081. (8) Chosrowjan, H.; Mataga, N.; Shibata, Y.; Tachibanaki, S.; Kandori, H.; Schichida, Y.; Okada, T.; Kouyama, T. J. Am. Chem. Soc. 1998, 120, 9706. (9) Kandori, H.; Furutani, Y.; Nishimura, S.; Schichida, Y.; Chosrowjan, H.; Shibata, Y.; Mataga, N. To be submitted (10) Chosrowjan, H.; Mataga, N.; Shibata, Y.; Imamoto, Y.; Tokunaga, F. J. Phys. Chem. B 1998, 102, 7695. (11) Mataga, N.; Chosrowjan, H.; Shibata, Y.; Imamoto, Y.; Tokunaga, F. J. Phys. Chem. B 2000, 104, 5191. (12) Rhodes, M. D.; Bennett, N.; Feeny, R. E. J. Biol. Chem. 1957, 234, 2054. (13) Tsuge, H.; Natsuaki, O.; Ohashi, K. J. Biochem. (Tokyo) 1975, 78, 835. (14) Gorelick, R. J.; Mizzer, J. P.; Thorpe, C. Biochemistry 1982, 21, 6936.

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10677 (15) Lau, S.-M.; Powell, P.; Buettner, H.; Ghisla, M.; Thorpe, C. Biochemistry 1986, 25, 4184. (16) Yagi, K.; Naoi, M.; Harada, M.; Okamura, K.; Hidaka, H.; Ozawa, T.; Kotaki, A. J. Biochem. (Tokyo) 1967, 61, 580. (17) Massey, V.; Swoboda, B. E. P. Biochem. Z. 1963, 338, 474. (18) Chosrowjan, H.; Mataga, N.; Nakashima, N.; Imamoto, Y.; Tokunaga, F. Chem. Phys. Lett. 1997, 270, 267. (19) Monaco, H. L. EMBO J. 1997, 16, 1475. (20) Kim, J.-J.; Wang, M.; Paschke, R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7523. (21) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153. (22) Mizutani, H.; Miyahara, I.; Hirotsu, K.; Nishina, Y.; Shiga, K.; Setoyama, C.; Miura, R. J. Biochem. (Tokyo) 1996, 120, 14. (23) Mattevi, A.; Vanoni, M. A.; Tonone, F.; Rizzi, M.; Teplyakov, A.; Coda, A.; Bolognesi, M.; Culti, B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7496. (24) Tanaka, F.; Tamai, N.; Yamazaki, I. Biochemistry 1989, 28, 4259. (25) Mataga, N.; Miyasaka, H. (a) Prog. React. Kinet. 1994, 19, 317. (b) AdV. Chem. Phys. 1999, 107, 431. (26) See for example, Bagchi, B.; Fleming, G. R. J. Phys. Chem. 1990, 94, 9. (27) See for example, (a) Mataga, N.; Ottolenghi, M. In Molecular Association, Vol. 2; Foster, R., Ed.; Academic Press: London, 1979; p 1. (b) Mataga, N. Pure Appl. Chem. 1984, 56, 1255. (c) Kakitani, T.; Yoshimori, A.; Mataga, N. In Electron Transfer in Inorganic, Organic, and Biological Systems, AdVances in Chemistry, Vol. 228; Bolton, J. R., Mataga, N., McLendon, G., Eds.; American Chemical Society, Washington, DC, 1991; Chapter 4. (d) Mataga, N. In Electron Transfer in Inorganic, Organic, and Biological Systems, AdVances in Chemistry, Vol. 228; Bolton, J. R., Mataga, N., McLendon, G., Eds.; American Chemical Society, Washington, DC, 1991; Chapter 6. (e) Kakitani, T.; Matsuda, N.; Yoshimori, A.; Mataga, N. Prog. React. Kinet. 1995, 20, 347. (28) (a) Mataga, N.; Tsuno, S. Naturwiss. 1956, 10, 305. (b) Mataga, N.; Tsuno, S. Bull. Chem. Soc. Jpn. 1957, 30, 711. (29) (a) Martin, M. M.; Ikeda, N.; Okada, T.; Mataga, N. J. Phys. Chem. 1982, 86, 4148. (b) Ikeda, N.; Miyasaka, H.; Okada, T.; Mataga, N. J. Am. Chem. Soc. 1983, 105, 5206. (c) Miyasaka, H.; Tabata, A.; Kamada, K.; Mataga, N. J. Am. Chem. Soc. 1993, 115, 7335. (d) Miyasaka, H.; Tabata, A.; Ojima, S.; Ikeda, N.; Mataga, N. J. Phys. Chem. 1993, 97, 8222. (30) (a) Mataga, N.; Migita, M.; Nishimura, T. J. Mol. Struct. 1978, 47, 199. (b) Okada, T.; Karaki, I.; Mataga, N. J. Am. Chem. Soc. 1982, 104, 7191. (31) Watenpaugh, K. D.; Sieker, L. C.; Jensen, L. H. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 3857 (32) See for example, (a) Kakitani, T.; Kimura, A.; Sumi, H. J. Phys. Chem. B 1999, 103, 3720. (b) Ando, K.; Sumi, H. J. Phys. Chem. B 1998, 102, 10991, and references therein.