J. Phys. Chem. 1986, 90,6833-6836
6833
One-Electron Oxidation of Flavins. A Flash Photolysis and Pulse Radlolysis Study P. F. Heelis,* B. J. Parsons, G. 0. Phillips, Research Division, North East Wales Institute, Deeside, Clwyd, CH5 4BR, United Kingdom
and A. J. Swallow Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, United Kingdom (Received: April 21, 1986; In Final Form: July 23, 1986)
One-electron oxidation of flavins in aqueous solution has been studied by using the reactions of the sulfate radical (SO4'-) and tetranitromethane with three different flavin derivatives in their ground and triplet excited states, respectively. The resulting spectra are compared and the results discussed in terms of the site of electron removal and the state of protonation of the resulting oxidized flavin radicals. It is concluded that electron loss occurs from the main *-electron system rather than a specific site and may be followed by deprotonation (pK, 6) in flavins unsubstituted at the N(3) position.
-
The photochemistry of flavins (I) (isoalloxazines) has been extensively studied, with a wide variety of both intermolecular and intramolecular photoreactions being reported.' Such phoF1
RI = R2 = RB = CHB, R4 = H Rl = R2 = RB = R4 = CHg isoalloxazine-N1O-butanoicR, = (CH2),COOH, R2 = RB= R4 = H acid lumiflavin 3-methyllumiflavin
toreactions have invariably been shown to proceed via either a one- or two-electron reduction of the flavin triplet state (3Fl).' In contrast, we recently reported2 an apparent one-electron oxidation of 3Fl in its reaction with tetranitromethane (eq 1). The
3Fl + C(NO2)4
- (no+) + F1"
C(N02);
+ NO2
(1)
properties of the flavin cation are of considerable interest for several reasons. Firstly, formation of the flavin cation in aqueous solution has been shown to occur via photoioni~ation.~ Secondly, the reactions of flavin triplet state(s) with another flavin molecule either in the ground state or a second triplet state have been suggested4 to occur via flavin-flavin electron transfer (eq 2), i.e., a D-D reaction analogous to that observed for many other dyestuffs. 3 ~ 1 +~i
-
FI*++ ~ 1 ' -
(2)
The second-order rate constant for triplet-ground-state quenching has been estimated as 3.7 X IO8 dm3 mol-' s-'.~ As the intrinsic rate constant5 of decay of the flavin triplet is approximately 670 s-I, then clearly a t the flavin concentrations typically employed ( 10-s-10-4 mol dm-3), reaction 2 could account for the deactivation of a significant proportion or even the majority of flavin triplet states. Therefore, the properties of the F1'+ radical need to be considered in interpreting the complex photochemical reactions of flavins. Indeed, the sub-uent reaction of Fl'+ with added substrates may in some cases4 account for substrate oxidation rather than direct attack by 3Fl. The flavin cation has also been detected in the flash photolysis of lumiflavin by using conductometric detection.6 In this case it was proposed to be formed upon excitation of a preformed flavin dimer (eq 3). In the present study, pulse radiolysis and flash 2F1* F12
- - hu
lF12
3F12
F1"
+ Fl'-
(3)
photolysis techniques are employed as independent methods of To whom correspondence should be addressed.
studying the spectral and other properties of Fl" in aqueous solution.
Experimental Section Lumiflavin was obtained from Sigma and purified chromatographically using a silica column and water/glacial acetic acid/l-butanol (5:1:4) as eluent. 3-Methyllumiflavin and isoalloxazine-N'o-butanoic acid were gifts from Prof. P. Hemmerich (Konstanz) and F. Muller (Wageningen), respectively. Tetranitromethane (Sigma) was washed 3 times with distilled water before use. All other chemicals were of Analar grade (B.D.H.) and were used as supplied. The solutions were made up in doubly distilled water and buffered (unless otherwise stated) with Na2HP04/KH2P04(pH 5-8, 0.01 mol dm-3). Solutions of pH < 5 or pH > 8 were obtained by addition of NaOH/HC104. All solutions were saturated with either oxygen-free nitrogen or nitrous oxide, with suitable precautions being taken to avoid unnecessary exposure to light. The pulse radiolysis experiments were carried out with a 814-MeV Vickers electron linear accelerator (pulse length 10 ns) as previously d e s ~ r i b e d . ~Typical ~ ~ experiments employed a 10-ns pulse with a radiation dose of 5.0 Gy/pulse. Optical cells of 2.5-cm path length were employed. Radiation doses were measured by using the absorption of (SCN)2'- formed by pulsing nitrous oxide saturated solutions of mol dm-3 KSCN taking Gc480= 46400 dm3 mol-' ~ m - ' . ~ The laser flash photolysis system was a JK Lasers System 2000 neodymium:YAG laser providing pulses of 20-11s duration at 353 nm as previously described.1° In both the flash photolysis and pulse radiolysis experiments, possible photolysis of the flavin solutions was avoided by using a continuous-flowsystem together with the use of appropriate lamp filters. Results Pulse radiolysis of nitrogen-saturated solutions of K2S208(0.005 mol dm-', pH 7) produced the characteristic spectrum of the SO4'(1) Heelis, P. F. Chem. SOC.Reu. 1982, 1 1 , 15. (2) Heelis, P. F.; Parsons, B. J.; Thomas, B.; Phillips, G. 0.J. Chem. Sac., Chem. Commun. 1985,954. ( 3 ) Getoff, N.; Solar, S.; McCormick, D. B. Science (Washington, D.C.) 1978, 201, 616. (4) Hemmerich, P.; Knappe, W. R.; Kramer, H. E. A.; Traber, R. Eur. J . Biochem. 1980, 104, 511. (5) Vaish, S. P.; Tollin, G. Bioenergetics 1970, 1 , 181. (6) Ballard, S. G.; Mauzerall, D. C.; Tollin, G. J . Phys. Chem. 1976.80, 341. ( 7 ) Keene, J. P. J . Sci. Instrum. 1964, 41, 493. (8) Heelis, P. F.; Parsons, B. J.; Phillips, G.0.;Land, E. J.; Swallow, A. J. J. Phys. Chem. 1982.86, 5169. (9) Schuler, R. H.; Patterson, L. K.; Janata, E. J . Phys. Chem. 1980, 84, 2088. (10) Heelis, P. F.; Phillips, G. 0. J . Phys. Chem. 1985, 89, 770.
0022-3654/86/2090-6833$01 .SO10 0 1986 American Chemical Society
The Journal of Physical Chemistry, Vol. 90, No. 26, 1986
6834
Heelis et al.
I
IO~AA
/
\ I'
iI.
-a
,
300
300
. ....:
i
500
400
600
700
h ( n m )
5b0
460
7100
660
(nm)
Figure 1. Spectra of the flavin cation derived from lumiflavin at pH 7, normalized at 640 nm. (A, -) Pulse radiolysis spectrum after the
reaction of SO4-, with the contributions from the reaction of tert-butanol radicals removed. (B, - - -) Flash photolysis spectrum after the reaction of the lumiflavin triplet state with tetranitromethane. (C, As for (B) after subtraction of the contribution of the nitroform radical (A 350-450
Figure 2. Spectra of the flavin cation derived from lumiflavin at pH 3.7, normalized to 640 nm. (A, -) Pulse radiolysis spectrum after the reaction of SO4'-, with the contribution from the reaction of tert-butanol radicals removed. (B, - - -) Flash photolysis spectrum after the reaction of the lumiflavin triplet state with tetranitromethane. (C, As for (B) after subtraction of the contribution of the nitroform radical. .-a)
-e)
nm).
radical" (A,= 450 nm) formed via the reaction of the hydrated electron with persulfate ions (eq 4). In the presence of lumiflavin
eaq-+ SzO&
-
SO4*-+ S04z-
(4)
(3 X lo-$mol dm-3) and tert-butyl alcohol as hydroxyl radical ) , decay of SO;- was more rapid scavenger (0.005mol d ~ n - ~the than in the absence of lumiflavin, and the concomitant buildup of a new species (Amx 640 nm) was observed. A rate constant of 7 X lo9dm3 mol-' s-' was determined for the reaction of SO4* with lumiflavin. However, pulse radiolysis of nitrous oxide saturated solutions of tert-butyl alcohol and lumiflavin (in the absence of persulfate ions) produced quite different spectra (A- 560 nm). In this case essentially all the primary radical species (OH', eq-) are converted to hydroxy-substituted tert-butyl (rert-butanol) radicals. Hence, it appears that tert-butanol free radicals also react with lumiflavin (a rate constant of 4 X lo9 dm3 mol-' s-l was determined). It should be noted that a relatively low concentration of tert-butyl alcohol was employed as it is known that SO4*-radicals react with the former." However, higher concentrations of terr-butyl alcohol (up to 0.1 mol dm-3) had no significant effect on the spectrum observed in nitrous oxide saturated solutions, thus demonstrating the complete scavenging of OH. In order to obtain the true spectrum of the product of the reaction of SO4'- with lumiflavin, the spectral contribution from the reaction of tert-butanol radicals with lumiflavin was subtracted from the observed spectrum in nitrogen saturated solutions (after taking due allowance of the differing G values for tert-butanol radical formation in nitrogen and nitrous oxide saturated solutions). These "subtraction" spectra are shown in Figure 1. An extinction coefficient at 640 nm of 3100 dm3 mol-l cm-' was also determined. The spectra in Figure 1 can be compared to that obtainedZ following the reaction of the triplet state of lumiflavin with tetranitromethane, also a t pH 7 (Figure 1B). As can be seen, the pulse radiolysis and flash photolysis spectra agree in the 430700-nm region. Below 430 nm, the strong absorption due to the nitroform radical (see eq 1) distorts the flash photolysis spectrum. An extinction coefficient of 3400 dm3 mol-' cm-l at 64Q nm was determined from the flash photolysis data (in good agreement to that obtained by using the pulse radiolysis technique) by reference to the extinction coefficient of the lumiflavin triplet state.'* By (11) Hayon, E.; Treinin, A.; Wilf, J. J . Am. Clrem. SOC.1972, 94, 47.
300
400
500
600
700
800
h(nm)
Figure 3. Absolute spectra of the flavin cation($ derived from lumiflavin at pH 7.0 (-) and 3.7 (---). Inset: The absorbance at 690 nm following the reaction of SO4'- with lumiflavin as a function of pH.
use of literature data on the nitroform radical,13 its contribution to the flash photolysis spectra was removed (Figure 1C)on the assumption that [Fl'+] = [C(NO,),-]. It is clear that the flash photolysis and pulse radiolysis spectra now broadly agree even in the 350-430-nm spectral region. At pH 3.7,an identical approach to that described at pH 7 was followed. Figure 2 shows the resulting spectra obtained by using pulse radiolysis and flash photolysis. Again, a good correspondence exists between the two spectra particularly after removal of the contribution from the nitroform radical absorption. However, the spectra obtained at pH 7 and 3.7,while similar, are significantly different, particularly in the long-wavelength region (A > 650 nm). Hence, the absorbance of the product of the reaction of SO4' with lumiflavin was studied by using pulse radiolysis as a function of pH. A clear pH dependence was observed (Figure 3 inset) from which a pK, of 6.0 f 0.3 can be derived. The absolute spectra of the lumiflavin cation(s) after correction for the ground-state spectrum are shown in Figure 3. In order to investigate the site of reaction on lumiflavin by SO4'-, flash photolysis of isoalloxazine-N'O-butanoic acid (IBA) was carried out at pH 7 in nitrogen-saturated solution. A transient ~
~~~~
(12) The previous estimate* of c6@ = 2300 dm' mol-' cm-' for F1'+ was determined by reference to a value of c6w = 4400 dm' mol-' cm-' for 'Fl. The
present estimate was obtained by use of a revised value of eJm = 3970 dm' mol-' cm-' for 'FI. This latter value was obtained by quantitative reduction of 'FI by a range of electron donors and subsequent measurement of the absorbance of the F1H' radical (esM) = 4400 dm' mol'' cm-l: Heelis, P. F., unpublished data). (13) Fujita, S.; Steenken, S . J . Am. Chem. SOC.1981, 103, 2540.
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