5694
J. Phys. Chem. B 2007, 111, 5694-5699
Donor-Acceptor Distance-Dependence of Photoinduced Electron-Transfer Rate in Flavoproteins Fumio Tanaka,*,† Haik Chosrowjan,‡ Seiji Taniguchi,‡ Noboru Mataga,‡ Kyosuke Sato,§ Yasuzo Nishina,§ and Kiyoshi Shiga§ SC1-414 Department of Chemistry, Faculty of Science, Mahasarakham UniVersity, Mahasarakham 44150, Thailand, Institute for Laser Technology, Utsubo-Hommachi 1-8-4, Nishiku, Osaka 550-0004, Japan, and Department of Molecular Physiology, Graduate School of Medical Sciences, Kumamoto UniVersity, Honjo 1-1-1, Kumamoto 860-8556, Japan ReceiVed: October 2, 2006; In Final Form: January 19, 2007
Ultrafast fluorescence quenching of flavin in flavodoxin from Megasphaera elsdenii was investigated by means of a fluorescence up-conversion method. Fluorescence lifetimes of flavodoxin from M. elsdenii were estimated to be τ1 ∼ 165 fs (0.97%) and τ2 ∼ 10 ps (0.03%). Correlation of photoinduced electron-transfer rates (kET) with averaged distances (Dav) between isoalloxazine and nearby tryptophan or tyrosine was examined and obtained an empirical equation of ln kET vs Dav by means of a nonlinear least-squares method using reported data together with flavodoxin from M. elsdenii. The values of Dav were calculated from X-ray structures of the flavoproteins. The ln kET was approximately linear at Dav shorter than 7 Å. The model free empirical equation was expressed as ln kET ) 29.7 + (-0.327Dav + 2.84 × 10-5)/(0.698 - Dav2). We also analyzed the observed values of ln kET with Marcus theory, but could not obtain reasonable results. Our analysis suggests that the average distance, rather than the shortest (edge to edge) distance or interplanar angles between the aromatics rings, is the key factor in the process of the photoinduced electron transfer in these flavoproteins.
Introduction Flavoproteins ubiquitously distribute in various microorganisms and in tissues such as the brains, kidneys, livers, and hearts of mammals, as well as in milk and leafy vegetables.1 These proteins play an essential role in oxidoreduction reactions.2 In some systems, flavins function as photoreceptors.3 The bright fluorescence of the flavins in an aqueous solution is quenched in the presence of aromatic amino acids, such as tryptophan (Trp) and tyrosine (Tyr).4-6 It was demonstrated by a transient absorption spectroscopy that the quenching process of flavin fluorescence can be ascribed to a photoinduced electron transfer (ET) from the Trp or Tyr to excited isoalloxazine (Iso) of flavins in solution7 and in flavoproteins.8 The ultrafast photophysics of various flavoproteins has also been investigated by means of a fluorescence up-conversion method.9,10 In these flavoproteins, Trp and Tyr are always located in the vicinity of the Iso ring. It has been shown with mutants of flavodoxin from DesulfoVibrio Vulgaris, strain Miyazaki, that both Trp and Tyr are effective quenchers for the fluorescence of Iso in the protein, but phenylalanine is not.11 Various microorganisms contain flavodoxins, which bind flavin mononucleotide (FMN) as the reaction center, and are considered to play an important role in electron transport reactions in these microorganisms.12 However, precise features of the oxidation-reduction reactions in proteins are still unclear. Flavodoxin may be classified into three groups with respect to the presence of Trp and Tyr near the Iso ring.12 Group 1 consists of flavodoxins in which Trp and Tyr sandwich the Iso ring; group 2, those in which Trp locates near Iso; and group 3, those * Corresponding author. E-mail:
[email protected]. † Mahasarakham University. ‡ Institute for Laser Technology. § Kumamoto University.
in which Tyr locates near the Iso ring.12 Flavodoxin from Megasphaera elsdenii belongs to group 2. Theories of ET13-21 have been tested with time-resolved fluorescence and transient absorption spectroscopy. The observed ET rates have been mostly analyzed with respect to the energy gap law of the theories.22-25 Another aspect of the theories, namely, donor-acceptor distance dependence of the ET rate, however, has rarely been reported. It is also important to verify the theories of ET with respect to the distance dependence. The experimental systems for it were difficult in bulk solution. Flavoproteins may be good models to test theories of the ET rate from the distance dependence, because in many flavoproteins, the distances between donors (Trp or Tyr) and acceptor (Iso) are known by X-ray crystallography. In the present work, we report our experimental results on ultrafast fluorescence dynamics of flavodoxin from M. elsdenii and analyses with a model free empirical equation and also Marcus ET theory13-15 of ln kET vs Dav in various flavoproteins,9-11 including flavodoxin from M. elsdenii. Methods Materials and Steady-State Measurements. Flavodoxin from M. elsdenii was prepared and purified according to the method described elsewhere.26 Absorption spectra were measured by a UV/VIS spectrophotometer (Jasco, Ubest-50), and fluorescence spectra by a fluorescence spectrophotometer (Hitachi, F-4500). Fluorescence Up-Conversion Technique: The femtosecond time-resolved fluorescence decay curves were measured using a fluorescence up-conversion apparatus. A Ti:sapphire laser system (Coherent, Inc. Verdi-V8 pumped Mira 900) was used as a light source (120 fs, 76 MHz, 800 mW at 820 nm). The pulses were further compressed up to ∼65 fs fwhm using a
10.1021/jp066450g CCC: $37.00 © 2007 American Chemical Society Published on Web 05/03/2007
Electron Transfer in Flavoproteins
J. Phys. Chem. B, Vol. 111, No. 20, 2007 5695
Figure 1. Chemical structures of isoalloxazine and aromatic amino acids and the corresponding atom notations. Figure 4. Parallel and perpendicular components of fluorescence decay dynamics of flavodoxin from M. elsdenii and its anisotropy measured at 530 nm.
Figure 2. Absorption and fluorescence spectra of flavodoxin from M. elsdenii.
Figure 3. Fluorescence decay dynamics of flavodoxin from M. elsdenii at 530 nm. The smooth line shows the biexponential fitting curve. The fitting residuals are shown in the upper panel of the Figure.
prism pair compressor. The second harmonic (∼20 mW) was generated in a 0.1-mm-thin BBO crystal and focused onto the sample circulating in a flow cell (50 mL/min) with 1 mm light path length to generate the fluorescence. It was collected with a pair of parabolic mirrors and focused together with the residual fundamental laser pulse on a 0.4 mm BBO type I crystal to generate the up-converted signal at the sum frequency. After passing through a grating monochromator (1200 g/mm, Acton Research Corp.), it was detected by a photomultiplier (R1527P) coupled with a photon counter (C5410) system (both from Hamamatsu Photonics K. K.). The fluorescence decay curves were obtained by varying the optical path length of the delay stage for the fundamental laser pulse. Ten scans (with 20 fs steps) in alternate directions were accumulated to give a single transient with acceptable signal-to-noise ratio. As an instrumental response function, a cross-correlation signal between fundamental and secondary harmonic pulses was used (fwhm ∼ 130 fs). All measurements were carried out at room
temperature (∼20 °C). In these experiments, the optical density per 1 cm path length was ∼3 at 410 nm. The method of anisotropy measurement was straightforward in fluorescence up-conversion experiments; namely, in the type I nonlinear crystal used in the experiments, only the fluorescence component parallel to the gate pulse was up-converted. Hence, for homogeneous media, when the excitation pulse was parallel to the gate pulse, the parallel component of the fluorescence (Ipara) was detected, and when the excitation pulse was perpendicular to the gate pulse, the perpendicular component (Iperp) was detected. Method of Analysis of Donor-Acceptor Distance-Dependent ET Rates. The distances between every atom of Iso and every atom of Trp or Tyr (chemical structures are shown in Figure 1) were calculated from the three-dimensional structures of flavoproteins determined by X-ray crystallography (coordinates of atoms were obtained from the Protein Data Bank). BASIC software was produced to calculate Dav, which was defined as the average of the distances of all the pairs between the atoms of the aromatic ring in Iso and the atoms of the aromatic ring in Trp or Tyr. The value of kET was evaluated as the inverse of the averaged lifetime, 1/∑iRiτi, where Ri and τi are the preexponential factor and lifetime of ith component, respectively, when a fluorescence decay function is expressed by a multiexponential function. In some flavoproteins, longer lifetime components were eliminated from the average lifetimes. The reason for this is described for each protein system in Table 2. First, we analyzed using various types of functions of ln kET vs Dav as empirical equations, which were expressed with several parameters. Among these functions, the following could reproduce the observed ln kET well,
ln kET ) d +
-aDav + b
(1)
c - Davm
where a, b, c, d, and m are unknown parameters to be determined. Second, we analyzed with the Marcus theory13-15, as eq 2,
[
kET ) ν0 exp -
{
-∆G0 -
e2 + λ0 + λs(Dav) 0Dav
4{λ0 + λs(Dav)}kBT
}
2
]
(2)
where ν0 is a frequency factor, -∆G0 is the standard free energy gap, and λ0 is the constant independent of solvent polarity. In
5696 J. Phys. Chem. B, Vol. 111, No. 20, 2007
Tanaka et al.
TABLE 1: Amino Acid Sequences of Four Flavodoxins Represented by One-Letter Symbols species
amino acid sequence
D. Vulgaris
55 L
G
A. nidulans
I
C. beijerinckii M. elsdenii
60 W
-
D
N
D 60 V
65 S
G
G
E
L
*
*
Y
M
G
D
-
-
*
*
M
G
S
60 E
E
L
*
*
L 85 L
S
T
G
C 55 C
P
W
L
G
C
L
G
C
S 55 P
T 55 A A
*
*
90 C
F
G
C
F 85 F
G
A
G 90 G
G
S
Y
F
G
S
Y
95 D
S
-
S
E
-
100 Y
G
Y 95 Y
D
Q
G 90 G
-
V 90 W
S
D
N
-
-
G
-
-
-
W
-
-
G
-
-
TABLE 2: Distances between Aromatic Substances and Isoalloxazine and Interplanar Angles of Aromatic Rings flavoprotein
atom in isoalloxazine
amino acid (interplanar angle with isoalloxazine)
atom in amino acid
distance (Å) Ds (Dav ( SE)
kETa (ps-1)
C8M C9a C8M C6 C8 C7 C8 C9a C8M C6 C4a O2 O2 C4a C10a
Trp-60 (34°) Tyr-98 (13°) Trp-57 (39°) Tyr-94 (4.2°) Trp-57 (48°) Tyr-94 (7°) Trp-57 (43°) Tyr-94 (9°) Trp-56 (40°) Tyr-98 (10°) Trp-90 (19°) Tyr-88 (65°) Tyr-88 (64°) Trp-90 (19°) Tyr-92 (3°) Tyr-224 (9°) Tyr-55 (47°) Benzoate (1°) Tyr-68 (77°) Trp-111 (9.5°) Trp-425 (73°) Tyr-515 (34°) Trp-166 (37°) Tyr-133 (32°) Tyr-375 (45°) Trp-156 (0°) Tyr-75 (0°)
CZ3 CE2 CZ3 O CZ3 OH CZ3 CE2 CZ3 O CZ2 CD2 CD2 CZ2 CD2
3.5 (6.4 ( 0.13) 3.3 (5.0 ( 0.10) 3.5 (6.5 ( 0.12) 3.3 (4.9 ( 0.10) 3.3 (6.4 ( 0.12) 3.3(4.8 ( 0.10) 3.3 (6.5 ( 0.12) 3.4 (4.8 ( 0.10) 3.3 (6.7 ( 0.13) 3.2 (4.9 ( 0.10) 3.3 (5.5 ( 0.12) 3.9 (10.1 ( 0.25) 3.9 (10.1 ( 0.25) 3.3 (5.5 ( 0.12) 3.5 (5.0 ( 0.11) 6.7 (8.1 ( 0.08) 7.3 (9.7 ( 0.12) 3.4 (5.3 ( 0.11) 3.6 (7.7 ( 0.16) 6.2 (12.2 ( 0.22) 7.0 (9.8 ( 0.10) 3.3 (8.3 ( 0.23) 3.9 (7.3 ( 0.14) 3.7 (8.8 ( 0.25) 4.7 (7.4 ( 0.13) 3.7 (3.7) 3.7 (3.7)
4.78b,11 3.73b,11
flavodoxin from D. Vulgaris28 (group 1) flavodoxin from A. nidulans29 (group 1) flavodoxin from E. coli31 (group 1) flavodoxin from Anabaena 712032 (group 1) flavodoxin from red algae33 (group 1) flavodoxin from C. beijerinckii30 WT (group 2) flavodoxin from C. beijerinckii G57T30 (group 2) flavodoxin from H. pylori34 (group 3) acid oxidase-benzoate complex35,36
D-amino
glucose oxidase37
medium-chain acyl-CoA dehydrogenase38 riboflavin binding protein39
6.06c
0.0222d 0.51710
2.4810 7.0410,11
n The values of kET were evaluated to be the inverse of the averaged lifetimes, ∑i)1 Riτi. When τi is longer g10 ps, which is considered to be a contribution of free flavins dissociated from the protein moieties during the measurements, it is eliminated in the averaging procedure. b Only shorter lifetimes were taken for kET, because quite an amount of FMN was considered to dissociate from the proteins during the circulation of the protein samples for the measurements. c The longer lifetime, ∼10 ps, in the present measurements originates from free FMN dissociated from the protein. d The lifetime (45 ps) of the dimer of D-amino acid oxidase40 (free from benzoate) was used for the kET estimation. a
the Marcus equation, the reorganization energy for the solvent, λs(Dav), is expressed as eq 3.13
(
λs(Dav) ) e2
)(
)
1 1 1 1 1 + 2a1 2a2 Dav ∞ 0
(3)
Here, e is electronic charge, a1 and a2 are the radii of the spherical ET acceptor and donor, and ∞ and 0 are the optical and static dielectric constants. e2/0Dav in eq 2 is the electrostatic energy. -∆G0 is expressed by the ionization potential of the donor, EIP, as follows,
-∆G0 ) -(G0 - EIP)
(4)
where G0 is the free energy related to the electron affinity of the acceptor. The values of EIP were 7.2 eV for Trp and 8.0 eV for Tyr.27 The radii of Iso, Trp, and Tyr were determined as follows: (1) The three-dimensional size of lumiflavin for Iso, 3-methylindole for Trp, and p-methylphenol for Tyr were obtained by MO calculations (PM3). (2) The volumes of these molecules were determined as asymmetric rotors. (3) The radii of spheres having the same volumes with asymmetric rotors were obtained.
The value of the a1 of Iso was 0.224 nm, and a2’s for Trp and Tyr were 0.196 and 0.173 nm, respectively. If the largest size of the asymmetric rotors was chosen as a1 and a2, then the value of λs became negative in riboflavin binding protein with R ) 0.37 nm. In the analysis with the Marcus theory, ν0, G0, λ0, and 0 were unknown. The unknown parameters were determined by a nonlinear least-squares method according to the Marquardt algorithm so as to obtain the minimum value of χ2 defined by eqs 5 and 6,
Dev(i) )
1
xY(i)
{YC(i) - Y(i)}
(5)
where the calculated and observed values of ln kET of the ith protein system are denoted by YC(i) and Y(i), respectively.
χ2 )
n
∑
1 Dev(i)2 n i)1
(6)
The number of protein systems, n, was seven. Results and Discussion Fluorescence Intensity and Anisotropy Decays of Flavodoxin from M. elsdenii. Absorption and fluorescence spectra
Electron Transfer in Flavoproteins of the flavodoxin are shown in Figure 2. Absorption peaks were at 377 and 446 nm, and emission was at 540 nm, which are similar to the spectral characteristics of other typical flavoproteins and FMN in aqueous solution. Fluorescence decay curve of flavodoxin at 530 nm is shown in Figure 3. It should be noted here that, similar to other flavoproteins,9-11 the dynamics of flavodoxin from M. elsdenii was independent of the wavelength of the emission monitored. The observed decay curve was analyzed with a sum of two exponential function, I(t) ) R1 exp(-t/τ1) + R2 exp(-t/τ2). The decay parameters were determined by a nonlinear least-squares method9-11 as τ1 ∼ 165 fs (R1 ) 0.97) and τ2 ∼ 10 ps (R2 ) 0.03), respectively. The shorter lifetime is similar to one of wild type flavodoxin from D. Vulgaris, 158 fs.11 The longer lifetime component may be ascribed to free FMN dissociated from the protein during circulation of the sample solution for the lifetime measurement. Fluorescence anisotropy is defined as (Ipara - Iperp)/((Ipara + 2Iperp), where Ipara and Iperp are polarized fluorescence intensities parallel and perpendicular to the polarization of the excitation laser pulse, respectively. The polarized fluorescence intensities, Ipara and Iperp, and anisotropy are shown in Figure 4. The values of the anisotropy in the time range examined (
