ETHYLCHLOROPHYLLIDE SENSITIZED PHOTOREDUCTION OF PHENOSAFRANINE
2633
Kinetics of the Ethyl Chlorophyllide Sensitized Photoreduction of Phenosafranine by Hydrazsbenzene
by G. R. Seely Contribution No. 181 from the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio (Received February 8, 1966)
~
~-
Ethyl chlorophyllide sensitizes the photoreduction of phenosafranine by hydrazobenzene and other reducing agents. According to the proposed mechanism, the reaction is initiated by transfer of an electron from triplet excited chlorophyllide to the dye, as was proposed earlier for the reduction of methyl red. Although evidently exergonic, because it can be made to occur in the dark, the reaction is subject to strong product retardation. This can be accounted for by the formation of a transient complex of the oxidized chlorophyllide radical with the reductant. This complex may decompose with oxidation of the reducing agent, thus completing the forward reaction, or it may react with leucophenosafranine, reversing the reaction and regenerating chlorophyllide. The direct, nonsensitized photoreduction of phenosafranine is not subject to such strong retardation. E.s.r. measurements of the concentration of phenosafranine semiquinone radicals suggest that these do not play a very important part in the retardation.
Introduction
Photochemical reactions of chlorophyll are of particular interest because of their probable involvement in photosynthesis. We have previously examined the photoreduction of the closely related ethyl chlorophyllide a by ascorbic acid in ethanol-pyridine solution& and the chlorophyllide-sensitized reduction of methyl red by ascorbic acid, hydrazobenzene, and mercaptosuccinic acid in ethanoL2 The two reactions were found to be quite unrelated : in the second, photoexcited chlorophyllide reacts with methyl red, not with the reductant, as in the first. The reduction of methyl red turned out to be kinetically very complex, apparently because unidentified reaction products affect the efficiencyof early steps. We wanted to examine a chlorophyllide-sensitized reduction in a system in which the dye could be recovered by oxidation, in the hope that side reactions would be less disturbing and the part played by chlorophyllide could be understood more clearly. The chosen oxidant was phenosafranine, a dye of low oxidation potential. Chlorophyll-sensitized reduction of related dyes, such as safranine T, has been The reducing agents effective for methyl red reduction
also worked for phenosafranine reduction; hydrazobenzene proved most convenient for detailed study even though the over-all reaction was not strictly reversible. Experimental The ethyl chlorophyllide a sample was prepared as before.'I2 Phenosafranine (Allied Chemical Corp.) was chromatographed on calcium carbonate and precipitated out of ethanol with cyclohexane; its extinction coefficient, in 1:6 ethanol :pyridine mixture, was 60,600 M-l cm.-l at 538 my. However, the rate of reduction was not sensitive to purification. Hydrazobenzene (Eastman) was recrystallized under nitrogen from ethanol until colorless. Stock solutions in ethanol were kept in a freezer. (1) G. R. Seely and A. Folkmanis, J . Am. Chem. SOC.,86, 2763 (1964). (2) G. R. Seely, J . Phys. Chem., 69,821 (1965). (3) T.T.Bannister, Photochem. PhotobioE., 2 , 519 (1963). (4) A. A. Krasnovskii, Dokl. A M . Nauk SSSR, 61, 91 (1948). (5) A. A. Erasnovskii and E. I(.Voinovskaya, ibid., 87, 109 (1952). (6) V. B. Evstigneev and V. A. Gavrilova, {bid., 98, 1017 (1954). (7) K. G. Mathai and E. Rabinowitch, J . Phys. Chem., 6 6 , 954 (1962).
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G. R. SEELY
2634
&Carotene (Eastman) was used as received. Ethanol was distilled from Mg(OCzH&, pyridine from BaO or AI2O3. The composition of the solvent, from pure ethanol to pure pyridine, had little influence on the rate of reduction; it was convenient to use a mixture of 1 part ethanol and 6 parts pyridine by volume. Quantum yields were determined as before, l r 2 by noting the progress of the reaction at intervals with a Beckman DU spectrophotometer and measuring light intensity with an Eppley thermopile. The reaction could also be followed continuously with a Cary Model 14 spectrophotometer, but quantum yields could not then be measured. Electron spin measurements were made with a Varian Model 4500 e.p.r. spectrometer, radical concentrations being estimated by comparison with known concentrations of diphenylpicrylhydrazyl. Samples were held in flat cells 0.34 mm. thick, which could also be fitted into the Cary spectrophotometer for concentration measurement. Light absorbed only by chlorophyllidewas isolated by a Baird-Atomic 660-mp interference filter; light absorbed by phenosafranine, with a Spectrolab 530-mp interference filter. The quantum yield was essentially independent of absorbed light intensity (usually in the neighborhood of einstein/l. sec.). Reactions were conducted under purified nitrogen’ at
the reduction of phenosafranine was almost equally rapid with either reductant. Sensitized reduction with hydrazobenzene is not reversible in the dark when the reductant is pure, and the chlorophyllide does not fade. These features made it the reductant of choice for quantum yield determinations. The reaction between hydrazobenzene and phenosafranine must be exergonic because it can be catalyzed in the dark by a strong base. With one phenosafranine preparation, an adventitious impurity also caused reduction in the dark. After all of these reactions, phenosafranine could be regenerated immediately and practically quantitatively upon exposure to air. In the dark, there was often a slow and incomplete recovery of the dye, dependent on the hydrazobenzene concentration. It was probably due to a persistent impurity in the hydrazobenzene. The rate of photoreduction was insensitive to the rate of the recovery in the dark. Dependence on Hydrazobenzene Concentration. The most notable difference between the reduction of pheno-
l.
(AH,), M 0
1.5x10-2
A
7.5~10-~
25O.
The following abbreviations are used: Chl is ethyl chlorophyllide a; D +, D , and DH are phenosafranine, its semiquinone, and leucophenosafranine, respectively; AH2, AH., and A are hydrazobenzene, its hydrazyl radical, and azobenzene, respectively.
Results Reducing Agents. Sensitized reduction of phenosafranine, with ascorbic acid or mercaptobenzothiazole as reductant, is spontaneously reversible in the dark; regeneration of phenosafranine is complete with a few minutes. In agreement with Bannister’s results with safranine T,3we found that with ascorbic acid a photostationary reduction level was established, proportional to the square root of the light intensity. The back reaction was approximately first order. With cysteine or mercaptosuccinic acid as reductant, the reaction was not reversible in the dark if oxygen was rigorously excluded. The chlorophyllide slowly faded during the reaction, apparently because of attack by mercaptyl radicals. Reduction went almost as fast as with hydrazobenzene. I n contrast to the reduction of methyl red, which was about 50 times faster with hydrazobenzene than with mercaptosuccinic acid,2 The Journal of Physical Chemistry
.
.\
t
10-41
I
I
I
I
I
I
1
ETHYLCHLOROPHYLLIDE SENSITIZED PHOTOREDUCTION OF PHENOSAFRANINE
safranine and that of methyl red is the occurrence in the former of strong retardation by the reduction product (Figure 1). Although a true photostationary state appears never to be reached, the retardation is so severe that the quantum yield is reduced to or l/loo of its initial value before the dye is half-reduced. A similar retardation was found in a run with ethyl pheophorbide a as the sensitizer. The retardation can be mitigated, but not overcome, by increasing the concentration of reductant. The initial quantum yield is hard to estimate, but it seems to approach 0.20 at high hydrazobenzene concentration. This is as high a value as any found for the reduction of methyl red.2 If the initial phenosafranine concentration, [D+IO, is decreased, the initial quantum yield is practically unchanged. The dependence of retardation on [D+IO suggests that the quantum yield depends on [DH] (or a quantity proportional to it) rather than on the ratio [DH]/[D+Io. The curves in Figure 1 have therefore been plotted with [DH] as the abscissa. Similar strong retardation was found in reduction of phenosafranine by cysteine or mercaptosuccinic acid. It is therefore not peculiar to hydrazobenzene and probably involves reduction products of the dye. Such retardation is rather unexpected for an exergonic reaction, and our experiments were directed partly toward elucidation of its cause. Nonsensitized Reduction. Unlike methyl red, phenosafranine does not require sensitization for its photoreduction.8 Runs were made in which phenosafranine alone absorbed light; some of the results are shown in Figure 2. The presence or absence of chlorophyllide does not affect the rate of this nonsensitized reduction. The initial quantum yield (40)approaches 0.40 at high reductant concentration, distinctly larger than for the sensitized reduction. It obeys the relationship
Retardation is still present but is not nearly so strong and does not prevent nearly complete reduction of the dye within a reasonable time. As shown by varying the initial phenosafranine concentration, the retardation in this case is a function of the ratio [D+]/[D+]o. Multiplication of observed quantum yields by [D+]o/[D+] largely compensates for the retardation, except, of course, in the run in which hydrazobenzene was deficient (see dotted curves of Figure 2). The retarder in the sensitized reduction must therefore affect steps involving the chlorophyllide. The initial quantum yield and the extent of retardation are independent of light intensity. The quantum
2635
Figure 2. Nonsensitized photoreduction of phenosafranine by hydrazobenzene. Semilogarithmic plot of quantum yield us. leucophenosafranine concentration, [DH]. Quantum yields multiplied by [D +]o/[D+] are shown by dashed lines. I n the first run (0),[D+]o = 7.5 x 10-6 M . I n the others, [D+JoE 1.3 X M . In the last run, hydrazobenzene is deficient.
yield is not affected by addition of FeC13 at 3.5 X M or of azobenzene at 1.7 X M . It was thought that the retardation might have something to do with the availability of protons, but added acetic acid a t 0.023 M had no effect. Strong bases (potassium or tetrabutylammonium hydroxide) convert phenosafranine into a form reduced by hydrazobenzene in the dark although the reaction is accelerated by light. Carotene Inhibition. 0-Carotene quenches the triplet state of c h l o r ~ p h y l l . ~From ~ ~ ~ the dependence on carotene concentration of the quantum yield of the sensitized reduction of methyl red by ascorbic acid, it was shown that chlorophyllide in the triplet state reacts with the oxidant rather than with the reductant.2 Similarly, the quantum yield of sensitized reduction of phenosafranine by hydrazobenzene depends on the carotene/phenosafranine ratio, rather than on the (8) G. I(. Oster, G. Oster, and C. Dobin, J . Phys. Chem., 66, 2511 (1962). (9) E.Fujimori and R. Livingston, Nature, 180, 1036 (1957). (10) H.Claes, 2.Naturforsch., 16b, 4.45 (1961).
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August 1966
G. R. SEELY
2636
i
(CAROTENE) /( Di), , 0
A
10-1
0.0 0.35
4
t
1.1 A
e
0 J
2.9 3.9
w>.
i: z
a
3 0
lo-2
I I0-31 0
A I I
1
I
1
I
I
I
1
2
3
4
5
6
7
IO6 ( D H ) , MOLAR
Figure 3. Sensitized photoreduction of phenosafranine by hydrazobenzene in the presence of p-carotene. Initial M , [AH21 = concentrations: [D+]o 1.3 X 4.4 x 10-4 M , [Chl] = 2 X M , I, = 0.9 X 10-6 einstein/l. sec. Compare also with fourth run of Figure 1.
carotenelhydrazobensene ratio. The quantum yields near the beginning of the runs in Figure 3 indicate M hydrazobenzene, the ratio of that with 4.4 X the rate constants of the reactions of triplet chlorophyllide with phenosafranine and p-carotene is about 0.7. Similar inhibition was found with 7.6 X 10-3 M hydrazobenzene. Since the bimolecular rate constant for the reaction of triplet chlorophyll with p-carotene is about 1.3 X lo9 M-’ sec.-l,gv10 the constant for its reaction with phenosafranine must be about 0.9 X lo9M-l sec.-le The curves in Figure 3 remain nearly parallel during the reaction; i.e., carotene has almost no effect on retardation. Electron Spin Resonance. We looked for radicals produced during the reduction of phenosafranine and the oxidation of hydrazobenzene that might be present in sufficient concentration to be important retarders. The e.s.r. spectrum of an acid form of the semiquinone of phenosafranine has been reported. l1 We detected a poorly resolved five-banded spectrum, apparently of a “neutral” form of the semiquinone, after chemical (dithionite) or photochemical reduction of phenosafranine in ethanol-pyridine mixtures. At The Journal of Physical Chemistry
ca. M phenosafranine and leucophenosafranine, the equilibrium constant for semiquinone formation K,
[D*I2/[D+][DH] (2) had the value 3 (*l) X However, the equilibrium constant unaccountably depended linearly on the leucophenosafranine concentration =
K,
=
4.5[DH]
(3)
At the phenosafranine concentrations used in the photoreduction experiments ([D+l0 n. 10-5 M ) the semiquinone concentration, according to (3), would not exceed -2 X lowsM . This low value is supported by our inability to detect intermediates spectrally during air oxidation of leucophenosafranine. The semiquinone concentration is probably too small to account for the retardation, according to the mechanism to be proposed. No signal could be detected when hydrazobenzene was partially oxidized by 12, FeC13, H202, or CuC12. When as little as 1% of the hydrazobenzene was oxidized with FeC13,the only product observed spectrally was azobenzene. There is therefore no reason to believe that any product other than azobenzene occurs, except as a transient, during the photochemical oxidation of hydraxobenzene.
Discussion Primary Reaction. The primary photochemical reaction is between photoexcited chlorophyllide and phenosafranine. This is shown by yields in the presence of carotene and is supported by other considerations. For example, the weak e.s.r. signal detected when a solution of chlorophyllide is illumiinated12 is trebled in the presence of phenosafranine but unaffected by hydrazobenzene Furthermore, hydrazobenzene is inactive as a reducing agent for chlorophyllide in ethanol-pyridine, unless an acid such as benzoic is added; even then the quantum yield for reduction of chlorophyllide is only about It is reasonable to suppose that the primary photochemical reaction is transfer of an electron from the triplet excited state of chlorophyllide (Chl*) to phenosafranine Chl*
+ D+-%
Chlf
+ D.
(4) The hypothesis that phenosafranine (or safranine T) has a low-lying triplet state to which it can be excited by energy transfer from ~hlorophyllide~ is not con(11) P.B. Ayscough and C. Thomson, J. Chem. SOC.,2055 (1962). (12) S. 8. Brody, G. Newell, and T.Castner, J. Phys. Chem., 64, 554 (1960).
ETHYLCHLOROPHYLLIDE SENSITIZED PHOTOREDUCTION OF PHENOSAFRANINE
sistent with differences in the degree of retardation of sensitized and nonsensitized reductions and the lack of an effect of carotene on retardation. Cause of Retardation. Before retardation in the sensitized reduction is explained, it is necessary to account for the much milder retardation of the nonsensitized reduction. A mechanism consistent with (1) begins with a primary photochemical reaction between photoexcited phenosafranine (D +*) and hydrazobenzene to form radicals. D+*
+ AH2+D* + AH. + H +
(5)
The radicals D . accumulate to a certain extent and equilibrate with D + and DH,13 but the radicals AH. are removed rapidly. The retardation can be adequately accounted for if AH. is removed by the two reactions
+ D+-%A + D. + H+ AH- + D H - % AH2 + D .
AH.
(6)
(7)
and the rate constants k2 and k3 are nearly equal. The second reaction is probable because we know from e.s.r. experiments that hydrazobenzene and D. do not react in the dark. As only (6) completes the reduction, a factor k2 [AH.1 [D+1/(h[AH.I [D+l
+
h[AH.l[DHI) G [D+l/[D+lo enters the expression for quantum yield. If this explanation is correct, the same factor should also apply to the sensitized reduction. However, multiplication of the quantum yield curves of Figure 1 by this factor accounts for only a small part of the retardation actually observed. The retarder does not compete with phenosafranine for Chl*, for, if it did, the curves of Figure 3 would converge rapidly as the reaction progressed, and the presence of carotene grow less important. The rate constant for the reaction between Chl* and the retarder would have to have an unreasonably large value -10’0 M-l set.-', and the retardation would depend on [DH]/ [D+] instead of on [DH]alone. The retarder does not merely compete with hydrazobenzene for reaction with Chl. + produced by (4) because retardation is not suppressed by sufficiently large [AH2]. On the other hand, retardation must precede the step by which AH. is formed, for after that the quantum yield is determined by the relative rates of (6) and ( 7 ) . The simplest way of accounting for the retardation
2637
is to assume that reduction of Chl. + by AH2 is not immediate upon.collision but is preceded by the formation of a relatively long-lived complex { Chl+.AH2}. Instead of decomposing to Chl and AH., this complex may react with the retarder, which now may be identiiied as DH. The radical D . may contribute to the retardation, but, in view of (3), its action would be hard to distinguish kinetically from that of DH. The proposed mechanism of chlorophyllide-sensitized reduction is therefore
C h l - 2 Chl* (8) where I , is the rate of light absorption (einstein/l. sec.) and a is the probability of conversion from excited singlet to triplet state. Chl* ko_ Chl
(9)
+ D + -% {Chl.+D.] {Chl-+D.}8 _ Chi.+ + D. {Chl.+D.]3 Chl + D+
Chl*
(10)
(11) (12)
where p is the probability that the radicals formed in the reaction “cage” designated by {Chl+.D } will diffuse away from each other.
-
+ DH-% Chi*+ + AH2
Chi.+
Chl
+ D. + H +
{Chl.+AH2]
(13) (14)
+ AH2 (15) {Chl*+AH2]-% Chl + AH. + H + (16) {Chl.+AH2}+ DH-% Chl + AH2 + De + H+ (17) {Chl-+AH2]-% Chi*+
The reaction is completed by steps 6 and 7 and the disproportionation 2D-
+ H + +D + + D H
(18)
Steps similar to (13) and (17) could be included with D in place of DH. A possible reaction between Chl* and DH, analogous to the reaction between Chl* and ascorbic acid’
Chl*
+ DH +ChlH- + D *
(19)
and its consequences have been omitted, but they might become important at high [DH]. With the usual steady-state approximations for all transient species the quantum yield becomes (13) The rate constant for dismutation of the similar semithionine radicals is 2.4 X 109 M-1 sec. -l, [C. G. Hatohard and C. A. Parker, Trans. Faraday sot., 57, 1093 (i961).]
Volum 69,Number 8 August 1966
G. R. SEELY
2638
Since ko is about 103/se~.,1~14-16 and kl is about lo9 M-I set.-', ko can be neglected in comparison with kl[D+] when [D+] is much above lov6 M . The first factor on the right side of (20) then reduces to ab, which is the initial quantum yield, 0.2. The third factor can be approximated by [D+J/[D+lo. As D H builds up and retardation becomes pronounced, k, becomes small compared with ks[DH]. Then, with the approximations of the preceding paragraph, (20) may be rearranged to a form asymptotically valid at high [DHJ
cup [D+l $
1
D+lo PHI -
-+
k~ k7
k4@a
+
k7)
kak.r[AHz]
+
k&s[DH] kslc7[AHz] (21)
When the left side of (21) is plotted against [DH], for runs shown in Figure 1 and for other runs also, the points do, in fact, fall on straight lines as [DH] becomes large, with slopes proportional to l/ [AHZ]. The intercepts at high [AH21 cluster about the value k8/k7 = lo6. The slopes of 11 runs with [AH21 ranging to 3.4 X lob5 M give the average from 1.6 X value k4/k5 = 200 i 40. At higher hydrazobenzene concentrations the slope is quite small, and the left side of (21) becomes practically independent of [DH1. Although the intercept tends to increase with decreasing [AH2], as expected from the second term on the right side of (21), the precision is not sufficient to permit an estimate of k6/k7. Its value, however, is surely less than 1and perhaps is nearly 0.
The Journal of Physical Chemistry
It is fairly safe to assume that k4 and ks are less than lo9 M-I set.-'. Then k, < 1 X lo3 set.-' and k5 < 5 X lo6 M-I set.-'. The complex { Chl+AH2] theresec. fore has a natural lifetime of a t least A complex of a similar sort must evidently be postulated for sensitized reductions with mercaptosuccinic acid and cysteine because these reactions are also retarded by product. It is not clear that such a longlived complex must exist in reductions with ascorbic acid; the reported3 similarity of quantum yields in the sensitized and unsensitized reactions argues against it. Apparently, that system is kinetically dominated by a rapid back reaction between the phenosafranine semiquinone and dehydroascorbic acid. An implication of the foregoing is that a long-lived complex is formed between Chl. + and those reducing agents that are not very labile, such as hydrazobenzene and the mercaptans, but not necessarily with ascorbic acid and leucophenosafranine. If such complexes exist as transitory intermediates during photosynthesis, they might be sites of action of inhibitors or stimulators. Certainly the existence of such complexes makes it easier to accept, if not to explain, the curiously varied effect of reaction products on the quantum yield of methyl red reduction.2 Specifically, { Chl+AH2] must be subject to attack by a transient and a final product, in the product-retarded photosensitized reduction of methyl red by hydrazobenzene. Acknowledgments. The author is indebted to Mr. D. Stoltz for technical assistance and to Mr. E. Brody for the electron spin resonance measurements, made under the direction of Mr. W. Treharne. The work was supported in part by National Science Foundation Grant No. GB-2089. (14) P.J. McCartin, Trans. Faradag Soc., 60, 1694 (1964). (15) R. Livingston and P. J. McCartin, J . Am. Chem. SOC.,85, 1671 (1963). (16) H.Linschitz and K. Sarkanen, ibid., SO, 4826 (1958).
