387
NEWS AND VIEWS
5 E. Ben-Hur, T. M. A. R. Dubbelman and J. Van Stevenchik, Phthalocyanine-induced photodynamic changes of cytoplasmic free calcium in Chinee hamster cells, Photochem. PhotobioZ., 54 (1991) 162166. 6 R. Wiegand-Steubing, S. Yeturu, A. Tuccillo, C.-H. Sun and M. W. Bems, Activation of macrophages by Photofrin II during photodynamic therapy, J. Phorochem. Photobiol. B: Biol., 10 (1991) 133-146. 7 T. Karu, N. Smolyaninova and A. Zelenin, Long-term and short-term responses of human lymphocytes to He-Ne laser radiation, Lasers Life Sci., 4 (1991) 167-178. 8 S. Young, P. Boulton, M. Dyson, W. Harwey and C. Diamatopoulos, Macrophage responsiveness to light therapy, Lasers Surg. Med., 9 (1989) 497-505. 9 A. C. Giese, Living with our Sun’s Ultraviolet Rays, Plenum, New York, 1976, p. 233. 10 T. Kant, Photobiological fundamentals of low-power laser therapy, IEEE J. Quantum Electron., 23 (1987) 1703-1717. 11 T. I. Karu, Molecular mechanism of the therapeutic effect of low-intensity laser radiation, Lasers Life Sci., 2 (1988) 53-74. 12 T. Karu, Effects of visible radiation on cultured cells, Photo&em. Photobiol., 52 (1990) 1089-1099. 13 R. J. Margolis, J. S. Dover, L. L. Polla, S. Watanabe, C. R. Shea, G. J. Hruza, J. A. Parrish and R. R. Anderson, Visible light action spectrum for melanin-specific selective photothermolysis, Lasers Surg. Med., 9 (1989) 389-397. 14 I. Freitas and G. F. Baronzio, Tumor hypoxia, reoxygenation and oxygenation strategies: possible role in photodynamic therapy, J. Photochem. Photobiol. B: Biol., II (1991) 3-30. 15 I. Freitas, Facing hypoxia: a must for photodynamic therapy, J. Photochem. Photobiol. B: Biol., 2 (1988) 281-282. 16 T. Karu, Photobiology of Low-Power Laser TheraW, Harwood Academic, London, Paris, 1989, p. 120. 17 J. A. Parrish and B. C. Wilson, Current and future trends in laser medicine, Photochem. Photobiol., 53 (1991) 729-730. 18 P. D. Kittlick, Inflammation, glycolytic metabolism, and glycosoaminoglycans, E~x Pathol., 30 (1986) 1-19.
Excited quartet states in DNA photolyase Paul F. Heelis Faculty of Science, Health and Medical Deeside, Clwyd, CH5 4BR (UK) Aziz
Studies,
The North
East
Wales Institute,
Connah’s
Quay,
Sancar
Department
of Biochemistry
and Biophysics,
University
of North
Carolina,
Chapel
Hill, NC 27599
(USA)
Tadashi Laboratory
Okamura of Physical
Chemistry,
Tokyo Zokei
University,
Hachioji,
Tokyo
193 (Japan)
1. Introduction DNAphotolyase (E.C.4.1.99.3) dimers in DNA into pyrimidines,
catalyzes the photochemical conversion of pyrimidine thus reversing the effect of far-UV (200-300 nm)
388
NEWS AND VIEWS
EmI TrpH
\
0
FADHe
Q
-
hu h>wonm
Q
T=lO.‘0s EM-
PTrpH
Back Reaction k=lO’s-’
4FADH*
“FADH*
0
\
T=lO%
Em-
/
FADH?
I
Trp*
1
0
k=e.g.lO’M-‘s-l
Rn
fir thmls
RS*
~~~~
FAD&
&~PH
Scheme 1. Photoreduction of the flavin radical of DNA photolyase. radiation. The enzyme from E. coli contains the flavin (FAD) blue neutral radical [l] and 5, lo-methenyltetrahydrofolate [2] as cofactors. Photoreactivation in vitro proceeds via the photoreduction of the flavin radical (FADHe) to give the fully reduced form (FADH,), followed by dimer repair by the photoexcited reduced form [3]. The folate acts in a secondary capacity as an antenna molecule, absorbing a photon and transferring energy to FADHz at the catalytic center. FADHz in turn transfers an electron to the pyrimidine dimer to generate a radical ion pair, resulting in dimer splitting. Our previous studies [4-71 using picosecond flash photolysis of DNA photolyase identified the primary excited state present 40 ps after excitation as the first excited doublet state of the flavin radical (Enz-*FADH . , 2, Scheme 1). Intersystem crossing to the lowest excited quartet state (Enz4FADH., 3) occurs within 100 ps. The intermediate Enz4FADH. then undergoes an internal electron (or hydrogen atom) transfer from a tryptophan (Trp) residue (3-4). Site specific mutagenesis has demonstrated that tryptophan-306 is the electron donor in this case [8]. Finally, either the primary reduction reaction is reversed over a period of tens of milliseconds (4+ 1) or, in the presence of extraneous electron donors, the back reaction is prevented by reduction of the Trp . (or TrpH+) radical (4 -+ 5) leading to the fully reduced enzyme (5). As the doublet and quartet excited states are novel intermediates in enzyme action, it would be of interest to discuss their properties further.
2. Properties
of flavin
radical
quartet
and doublet
states
The familiar concepts of orbital classification (e.g. n?r*, nn*) and spin multiplicity are equally applicable to radical excited states. However, as a consequence of the presence of an unpaired electron, we need to consider doublet and quartet states instead of singlet and triplets. Probable electronic configurations of the flavin radical excited states are shown in Scheme 2. Physicists have long looked for the excited quartet states of organic radicals. However, due to the fact that the lowest energy quartet state (a,,) is normally expected to be higher in energy than the lowest energy doublet state (Dr), intersystem crossing is unlikely. The only energetically feasible route for intersystem crossing is from a higher energy doublet state (D2 +a,). However, kinetically this is unlikely, due to the high rate of the competing internal conversion (D2 + D,). Nonetheless, an electron spin echo study has suggested that short-wavelength excitation of the benzophenone ketyl radical leads to such a process [9].
NEWS AND VIEWS
rI
rI
n
I---I-
+-I-
GROUND STATE DOUBLET
hv ___)
Scheme 2. Possible
Scheme 3. Proposed
EXCITED STATE DOUBLET
electronic
Jablonski
_ _‘_“f: _ +
configuration
diagram
4 Q&lll”‘)
J),(IIII*)
Do
389
EXCITED STATE QUARTET
of TIT* doublet
and n?r* quartet
showing D,(vrr*) + Q,(n?S)
states.
intersystem
crossing.
In the case of DNA photolyase, laser excitation of Enz-FADH. at either 355 or 530 nm generates the same transient species with the same quantum yield [4, 51; thus intersystem crossing from a higher energy doublet state is clearly not involved. In the case of the flavin radical, excitation in the longest wavelength band 500-700 nm almost certainly involves arrr* transitions. We propose that the presence of a low-lying quartet state of nv* character provides the route for intersystem crossing. Spin-orbit coupling of Di(m*) and Q,(na) (Scheme 3) would enhance the possibility of intersystem crossing competing with internal conversion. The excited doublet has a lifetime of approximately 100 ps which is similar to that of many other organic doublet states [lo]. The much longer quartet lifetime of 1 ps is consistent with the spin-forbidden nature of the intersystem crossing from Q,, to Da. The only other reported evidence of an excited quartet is the phosphorescence emission from the decacyclene anion for which ~=2.6 ms at 77 K [ll]. The quartet reacts in wild-type photolyase with tryptophan-306 via electron transfer. If tryptophan-306 is substituted by phenylalanine, the quartet lifetime does not change appreciably ( f 20%) suggesting that k(electron transfer) -=~/ck(Q,, + Do). @ (electron transfer) is known to be 0.1, and thus it follows that @(Qo)>>O.l. We have also studied another flavoprotein radical derived from glucose oxidase enzyme and have found that a very similar excited quartet state is also formed in this case.
390
NEWS AND VIEWS
References 1 M. S. Joms, G. B. Sancar and A. Sancar, Identification of a neutral flavin radical and characterization of a second chromophore in E. coli DNA photolyase, Biochemistry, 23 (1984) 1856-1861. 2 J. L. Johnson, S. Hamm-Alvarez, G. Payne, G. B. Sancar, K. V. Rajagopalan and A. Sancar, Identification of the second chromophore of Escherichiu coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate, fioc. Natl. Acad. Sci. USA, 85 (1988) 2046-2050. 3 G. Payne, P. F. Heelis, B. R. Rohrs and A. Sancar, The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro, Biochemism, 26 (1987) 7121-7127. 4 P. F. Heelis and A. Sancar, Photochemical properties of E. coli DNA photolyase. A flash photolysis study, Biochemidy, 25 (1986) 8163-8166. 5 P. F. Heelis, G. Payne and A. Sancar, Photochemical properties of E. coli, DNA photolyase: selective photodecomposition of the second chromophore, Biochemistry, 27 (1987) 4634-4640. 6 P. F. Heelis, T. Okamura and A. Sancar, Excited state properties of Escherichia coli DNA photolyase in the picosecond to millisecond range, Biochemistv, 29 (1990) 5694. 7 T. Okamura, A. Sancar, P. F. Heelis, Y. Hirata and N. Mataga, Doublet+uartet intersystem crossing of flavin radical in DNA photolyase, 1 Am. Chem. Sot., 121 (1989) 5967. 8 Y. F. Li, P. F. Heelis and A. Sancar, Active site of DNA photolyase: Tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro, Biochemkhy, 30 (1991) 6322-6329. 9 M. C. Thurnauer and D. Meisal, Time resolved EPR studies of benzophenone-diphenyl ketyl radical. Possible evidence for quartet-doublet intersystem crossing, Chem. Phys. Lett., 92 (1982) 343-345. 10 V. A. Smirnov and V. G. Plotnikov, The luminescence spectroscopic properties of aromatic radicals and biradicals, Russ. Chem. Rev., 55 (1986) 929-947. 11 C. J. M. Brugman, R. P. H. Rettschinck and G. J. Hoytink, Quartet-doublet intersystem crossing from an aromatic radical, Chem. Phys. Lett., 8 (1971) 263-264.
NEWS AND VIEWS
5 E. Ben-Hur, T. M. A. R. Dubbelman and J. Van Stevenchik, Phthalocyanine-induced photodynamic changes of cytoplasmic free calcium in Chinee hamster cells, Photochem. PhotobioZ., 54 (1991) 162166. 6 R. Wiegand-Steubing, S. Yeturu, A. Tuccillo, C.-H. Sun and M. W. Bems, Activation of macrophages by Photofrin II during photodynamic therapy, J. Phorochem. Photobiol. B: Biol., 10 (1991) 133-146. 7 T. Karu, N. Smolyaninova and A. Zelenin, Long-term and short-term responses of human lymphocytes to He-Ne laser radiation, Lasers Life Sci., 4 (1991) 167-178. 8 S. Young, P. Boulton, M. Dyson, W. Harwey and C. Diamatopoulos, Macrophage responsiveness to light therapy, Lasers Surg. Med., 9 (1989) 497-505. 9 A. C. Giese, Living with our Sun’s Ultraviolet Rays, Plenum, New York, 1976, p. 233. 10 T. Kant, Photobiological fundamentals of low-power laser therapy, IEEE J. Quantum Electron., 23 (1987) 1703-1717. 11 T. I. Karu, Molecular mechanism of the therapeutic effect of low-intensity laser radiation, Lasers Life Sci., 2 (1988) 53-74. 12 T. Karu, Effects of visible radiation on cultured cells, Photo&em. Photobiol., 52 (1990) 1089-1099. 13 R. J. Margolis, J. S. Dover, L. L. Polla, S. Watanabe, C. R. Shea, G. J. Hruza, J. A. Parrish and R. R. Anderson, Visible light action spectrum for melanin-specific selective photothermolysis, Lasers Surg. Med., 9 (1989) 389-397. 14 I. Freitas and G. F. Baronzio, Tumor hypoxia, reoxygenation and oxygenation strategies: possible role in photodynamic therapy, J. Photochem. Photobiol. B: Biol., II (1991) 3-30. 15 I. Freitas, Facing hypoxia: a must for photodynamic therapy, J. Photochem. Photobiol. B: Biol., 2 (1988) 281-282. 16 T. Karu, Photobiology of Low-Power Laser TheraW, Harwood Academic, London, Paris, 1989, p. 120. 17 J. A. Parrish and B. C. Wilson, Current and future trends in laser medicine, Photochem. Photobiol., 53 (1991) 729-730. 18 P. D. Kittlick, Inflammation, glycolytic metabolism, and glycosoaminoglycans, E~x Pathol., 30 (1986) 1-19.
Excited quartet states in DNA photolyase Paul F. Heelis Faculty of Science, Health and Medical Deeside, Clwyd, CH5 4BR (UK) Aziz
Studies,
The North
East
Wales Institute,
Connah’s
Quay,
Sancar
Department
of Biochemistry
and Biophysics,
University
of North
Carolina,
Chapel
Hill, NC 27599
(USA)
Tadashi Laboratory
Okamura of Physical
Chemistry,
Tokyo Zokei
University,
Hachioji,
Tokyo
193 (Japan)
1. Introduction DNAphotolyase (E.C.4.1.99.3) dimers in DNA into pyrimidines,
catalyzes the photochemical conversion of pyrimidine thus reversing the effect of far-UV (200-300 nm)
388
NEWS AND VIEWS
EmI TrpH
\
0
FADHe
Q
-
hu h>wonm
Q
T=lO.‘0s EM-
PTrpH
Back Reaction k=lO’s-’
4FADH*
“FADH*
0
\
T=lO%
Em-
/
FADH?
I
Trp*
1
0
k=e.g.lO’M-‘s-l
Rn
fir thmls
RS*
~~~~
FAD&
&~PH
Scheme 1. Photoreduction of the flavin radical of DNA photolyase. radiation. The enzyme from E. coli contains the flavin (FAD) blue neutral radical [l] and 5, lo-methenyltetrahydrofolate [2] as cofactors. Photoreactivation in vitro proceeds via the photoreduction of the flavin radical (FADHe) to give the fully reduced form (FADH,), followed by dimer repair by the photoexcited reduced form [3]. The folate acts in a secondary capacity as an antenna molecule, absorbing a photon and transferring energy to FADHz at the catalytic center. FADHz in turn transfers an electron to the pyrimidine dimer to generate a radical ion pair, resulting in dimer splitting. Our previous studies [4-71 using picosecond flash photolysis of DNA photolyase identified the primary excited state present 40 ps after excitation as the first excited doublet state of the flavin radical (Enz-*FADH . , 2, Scheme 1). Intersystem crossing to the lowest excited quartet state (Enz4FADH., 3) occurs within 100 ps. The intermediate Enz4FADH. then undergoes an internal electron (or hydrogen atom) transfer from a tryptophan (Trp) residue (3-4). Site specific mutagenesis has demonstrated that tryptophan-306 is the electron donor in this case [8]. Finally, either the primary reduction reaction is reversed over a period of tens of milliseconds (4+ 1) or, in the presence of extraneous electron donors, the back reaction is prevented by reduction of the Trp . (or TrpH+) radical (4 -+ 5) leading to the fully reduced enzyme (5). As the doublet and quartet excited states are novel intermediates in enzyme action, it would be of interest to discuss their properties further.
2. Properties
of flavin
radical
quartet
and doublet
states
The familiar concepts of orbital classification (e.g. n?r*, nn*) and spin multiplicity are equally applicable to radical excited states. However, as a consequence of the presence of an unpaired electron, we need to consider doublet and quartet states instead of singlet and triplets. Probable electronic configurations of the flavin radical excited states are shown in Scheme 2. Physicists have long looked for the excited quartet states of organic radicals. However, due to the fact that the lowest energy quartet state (a,,) is normally expected to be higher in energy than the lowest energy doublet state (Dr), intersystem crossing is unlikely. The only energetically feasible route for intersystem crossing is from a higher energy doublet state (D2 +a,). However, kinetically this is unlikely, due to the high rate of the competing internal conversion (D2 + D,). Nonetheless, an electron spin echo study has suggested that short-wavelength excitation of the benzophenone ketyl radical leads to such a process [9].
NEWS AND VIEWS
rI
rI
n
I---I-
+-I-
GROUND STATE DOUBLET
hv ___)
Scheme 2. Possible
Scheme 3. Proposed
EXCITED STATE DOUBLET
electronic
Jablonski
_ _‘_“f: _ +
configuration
diagram
4 Q&lll”‘)
J),(IIII*)
Do
389
EXCITED STATE QUARTET
of TIT* doublet
and n?r* quartet
showing D,(vrr*) + Q,(n?S)
states.
intersystem
crossing.
In the case of DNA photolyase, laser excitation of Enz-FADH. at either 355 or 530 nm generates the same transient species with the same quantum yield [4, 51; thus intersystem crossing from a higher energy doublet state is clearly not involved. In the case of the flavin radical, excitation in the longest wavelength band 500-700 nm almost certainly involves arrr* transitions. We propose that the presence of a low-lying quartet state of nv* character provides the route for intersystem crossing. Spin-orbit coupling of Di(m*) and Q,(na) (Scheme 3) would enhance the possibility of intersystem crossing competing with internal conversion. The excited doublet has a lifetime of approximately 100 ps which is similar to that of many other organic doublet states [lo]. The much longer quartet lifetime of 1 ps is consistent with the spin-forbidden nature of the intersystem crossing from Q,, to Da. The only other reported evidence of an excited quartet is the phosphorescence emission from the decacyclene anion for which ~=2.6 ms at 77 K [ll]. The quartet reacts in wild-type photolyase with tryptophan-306 via electron transfer. If tryptophan-306 is substituted by phenylalanine, the quartet lifetime does not change appreciably ( f 20%) suggesting that k(electron transfer) -=~/ck(Q,, + Do). @ (electron transfer) is known to be 0.1, and thus it follows that @(Qo)>>O.l. We have also studied another flavoprotein radical derived from glucose oxidase enzyme and have found that a very similar excited quartet state is also formed in this case.
390
NEWS AND VIEWS
References 1 M. S. Joms, G. B. Sancar and A. Sancar, Identification of a neutral flavin radical and characterization of a second chromophore in E. coli DNA photolyase, Biochemistry, 23 (1984) 1856-1861. 2 J. L. Johnson, S. Hamm-Alvarez, G. Payne, G. B. Sancar, K. V. Rajagopalan and A. Sancar, Identification of the second chromophore of Escherichiu coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate, fioc. Natl. Acad. Sci. USA, 85 (1988) 2046-2050. 3 G. Payne, P. F. Heelis, B. R. Rohrs and A. Sancar, The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro, Biochemism, 26 (1987) 7121-7127. 4 P. F. Heelis and A. Sancar, Photochemical properties of E. coli DNA photolyase. A flash photolysis study, Biochemidy, 25 (1986) 8163-8166. 5 P. F. Heelis, G. Payne and A. Sancar, Photochemical properties of E. coli, DNA photolyase: selective photodecomposition of the second chromophore, Biochemistry, 27 (1987) 4634-4640. 6 P. F. Heelis, T. Okamura and A. Sancar, Excited state properties of Escherichia coli DNA photolyase in the picosecond to millisecond range, Biochemistv, 29 (1990) 5694. 7 T. Okamura, A. Sancar, P. F. Heelis, Y. Hirata and N. Mataga, Doublet+uartet intersystem crossing of flavin radical in DNA photolyase, 1 Am. Chem. Sot., 121 (1989) 5967. 8 Y. F. Li, P. F. Heelis and A. Sancar, Active site of DNA photolyase: Tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro, Biochemkhy, 30 (1991) 6322-6329. 9 M. C. Thurnauer and D. Meisal, Time resolved EPR studies of benzophenone-diphenyl ketyl radical. Possible evidence for quartet-doublet intersystem crossing, Chem. Phys. Lett., 92 (1982) 343-345. 10 V. A. Smirnov and V. G. Plotnikov, The luminescence spectroscopic properties of aromatic radicals and biradicals, Russ. Chem. Rev., 55 (1986) 929-947. 11 C. J. M. Brugman, R. P. H. Rettschinck and G. J. Hoytink, Quartet-doublet intersystem crossing from an aromatic radical, Chem. Phys. Lett., 8 (1971) 263-264.
