Further studies of one electron reduction of 1,10-phenanthroline-5,6-quinone in aqueous solutions

Radiat. Phys. Chem. Vol. 46, No. 4-6, pp. 545-548, 1995 Copyright © 1995 Elsevier Science Ltd 0969-806X(95)00214-6 Printedin Great Britain. All rights reserved 0969-806X/95 $9.50 + 0.00

Pergamon

FURTHER STUDIES OF ONE ELECTRON REDUCTION OF 1,10PHENANTHROLINE-5,6-QUINONE IN AQUEOUS SOLUTIONS H. Bao*, S. Navaratnam**, B. J. Parsons** and G. O. Phillips**

* Department o f Chemistry, Beij'ing Normal University, Beijing, 100875, PRC; and ** Multidisciplinary Research and Innovation Center. Faculty o f Science, Health and Medical Studies. The North East Wales Institute, Deeside, Clwyd, CH5 4BR, UK

ABSTRACT One-electron reduction of 1,10-phenanthroline-5,6-quinone (PQ) in aqueous solutions was studied by pulse radiolysis. It was found that the initial transient species formed by the reaction of eaq- with PQ at neutral pH has an absorption spectrum with maxima at 365 nm and 480 nm. The decay of the species follows a first order kinetics to form a subsequent transient species with an absorption maxirnurn at 490 nm. Similar changes were also observed at pH 4.1. The formation and decay kinetics of both initial and subsequent transients at various pI-Is were investigated. The ionic strength has no effect on the decay of the subsequent transient at 4.1. On the other hand, the decay at neutral pHs was accelerated dramatically with increasing ionic strength. The plot according to Bronsted Bjerrum equation gives a slope of--4.

KEYWORBS Pulse radiolysis; 1,10-phenanthroline-5,6-quinone; one-electron reduction; lipoxygenase

~TRODUCTION Lipoxygenase is a non-Mean iron-containing enzyme, which catalyses the dioxygeuation of polyunsaturated fatty acid to form fatty acid hydroperoxides. It is possible that an orth~uinone, pyrroloquinoline quinone (PQQ) is involved in the catalysis through its one-electron reduced form, serrfiquinone radical, causing iron to cycle between the Fe(ll) and Fe(EI) states (van der Meer et al., 1988). To elucidate the possible pathway for one-electron transfer in the enzyme, we have studied several model compounds for PQQ and lipoxygenase (Bao, 1993; Bao et al., 1992). PQ is selected since it has a structure similar to PQQ. In addition, PQ is a good chaletor and can form complexes with several metals (Goss et al., 1985). This property enables an easy preparation of iron-PQ complexes and thereby the study of a good model complex for the lipoxygenase is made feasible. The preliminary studies of one-electron reduction of PQ were presented in several meetings (Bao et al., 1992; Navaratnam et al., 1989). A more detailed study is presented in this paper.

EXPERIMENTAL PQ was synthesed from 1,10-phenanthroline (Smith et al., 1947; Kofl et al., 1962; Dickeson et aL, 1970). The "spectrosol" solvent isopropanol was purchased from Romil Chem. Ltd. The HPLC grade t-butanol was supplied by Fluka Chem. Co. "White spot" N 2 (>99.99%) and N20 (atomic absorption grade, >99.9%) were supplied by B. O. C. Ltd. All other reagents used are at least of analytical purity. The pulse radiotysis experiments were carded out with a 8-14 MeV Vickers electron linear accelerator as previously described (Bao et al., 1993) using quartz cells with path lengths of 1.5 and 2.5 era. Radiation doses were established using air-saturated lxl0 -2 mol dm"3 aqueous of KSCN and taking G[(SCN)2e-]. e = 2.23x10 "4 m 2 j-1 cm-I (Fielden, 1982).

545

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H. Bao e t al. RESULTS AND DISCUSSION

Reduction by Hydrated Electron On pulsing a N2-saturated aqueous solution containing PQ and t-butanol at pH 7.9, all the eaq- generated decayed rapidly within - 500 ns by reacting with PQ resulting in absorption changes in the UV and visible regions. The decay rate of the absorption at 720 nm due to the eaq- agrees with the formation rate at-365 m due to an initial product. From a plot of the pseudo-first-order rate constant at 720 nm against the concentration of PQ, the bimolecular rate constant for the reaction of eaq" with PQ was determined to be (3.2i-0.2)×1010 dm 3 mo1-1 s-1. This value is pH independent between pI-Is 4.7-9.6. Fig.la shows the uncorrected transient absorption spectra at different times obtained under this case. The initial spectrum shows a broad band with a ~ m x at 480 nm and a small band centered at 365 nm. The band centered at 365 nm decayed with a concomitant increase in the visible region. After 8 ~ , the absorption shows raaxima at 490 nm and below 320 rim. Similar changes in absorption were also observed at pH 4.1 (Fig. lb). However, both the initial and the subsequent absorption in the visible region are centered at longer wavelengths. The decay of the initial transient monitored at 365 nm was found to be relatively fast and followed first-order kinetics with a rate constant of (1.2x~0.2)×105 s-1, which is independent pH over the range of 5.8 to 9.5. Below pH -5, the second-order decay of the subsequent absorption becomes much faster, which made the analysis of the decay difficult. However, comparing the fLrst-order build-up oftha absorption at 490 ran with that at nentral pH, it was suggested that the first-order decay rate constant at low pHs is not different from that at neutral ~H and is also in the order of 105. ol8

@.06 m

_s

. e

I

-

J.

..

0.@1 koo aao Wavelength

(nm)

J

i

i

wavelength ( n m )

Fig. I. Uncorrected absorption spectra obtained after giving a 27.9 Gy electron pulse to N2-saturated aqueous solutions of 2.0xl0 -4 mol dm-3 PQ in the presence of 0.5 tool dm-3 t-butanol and Ixl0 "2 tool dm-3 phosphate, 1.5 cm cell (a) pH 7.9, 1.0 ~s (O), 8.2 p~ (o) and 18 ~ (A); (b) pH 4.1, 0.98 ~s (O), 9.0 gs (o) and 18 gs (A) Reduction by Isopropanol Radical The corrected absorption spectra of the transient(s) obtained at the end of the pulse due to the reaction of (CH 3)2~OH with PQ at pH 7.5 and 3.8 are shown in Fig. 2. They are identical to those caused by CO2"- reaction (Bao, 1992). The molar extinction coefficients at 490 ran (at pH7.5) and at 510 nm (at pH 3.8) were found to be 4000-2400 dm 3 tool-1 cm-1 and 3500+500 dm3 tool-l an -1 respectively. Clearly, the reduction of PQ by (CH3)2~OH radicals produces the same species as those by C O 2 " i.e. a 490 nm species at pH 7.5 and a 510 nm species at pH 3.8. These two species are not the initial species observed in the reaction of eaq- with PQ but are suggested to be the same as the subsequent transient species formed by eaqwith PQ at similar pHs. The disappearance of the 490 nm and 510 nm species was found to obey second-order kinetics over pHs range of 2.6 to 9.0. Fig. 3 shows the plot of 2k vs pH. Between pHs 3.5-5.0, the value of 2k is 1.2xi09+I0% dm 3 tool-l s-l. At pH>5, the decay rate constant starts to decrease quickly until pH -6.5. At pH>6.5, the decrease of the 2k is smaller. Furthermore at pH>7, the absorption at --490 nm did not disappear completely, a residual absorption remained constant for several milliseconds, and this residual absorption increased with increasing pH. A similar observation was made for the decay of the species produced by eaq-. This may indicate that at high pHs the 490 nm species decays to form different products compared to that at pH 3.5-5.0. In order to ascertain the charge of the 490 nm and 510 nm species, the ionic strength effect on the decay of the absorption at these two wavelengths was investigated in the presence of known amounts of sodium perchlorate at pH - 7 and --4, respectively. At pH 4, it was found that the ionic strength has no effect on the decay of the absorption which indicates the

9th International Meeting on Radiation Processing

547

510 run species is a neutral radical. On the other hand, the decay at pH-7 was accelerated dramatically with increasing ionic strength. From a plot of log2k against "~I/(1+4) acx~rding to the Br0nsted-Bjerrum equation (1), a slope of 3.9 was derived (Fig. 4). This value implies either beth species have a charge of 2 or one of them has a charge of 4 and the other 1. log2k=-log2ko + 1.02 Za ZB[~I/(I+a~I)] (1) where, 2k and 2ko are the 2nd-order rate constants at ionic strength of I and O, respectively, ZA and Z B are charges of the ions and ct is a constant taken to be 1. It was also found that unlike the decay of the initial absorption at 365 nm, the decay of the subsequent absorption at 490 nm increased linearly with phosphate ion concentration (Fig 5). 8000

16

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T710 14 12

4000

•

s

20OO

6

o

tO

0 2

300

400

500

600

3

Fig. 2. Corrected absorption spectra obtained at 13 gs alter giving a 14.6 Gy electron pulse to N20saturated solutions containing lx10.4 mol dm-3 PQ and 0.5 mol dm-3 isopropanol at pH 7.5 (e) and 3.8 ( ,).

5

6

7

8

9

Fig. 3. Dependence of the 2nd-order decay rate constant on pH observed at 490 nm. Pulse radiolysis of N20-saturated solutions containing PQ, isopropanol and phosphate buffer (lxl0 "4, 0.5 and 5x10.4 mol dm-3, respectively). I=0.1mol dm "3, Dose=8.4 Gy.

8

8.4 8.2 8

7.8

2

7.6 7.4

7.2

4

pH

wavelength (nm)

|

i

i

0.1

0.2

0.3

~I](l+~I)

Fig. 4. Dependence of the 2nd-order decay rate constant on ionic strength (observed at 490 nm). Pulse radiolysis of N20-saturated solutions containing 9.3x10 "5 mol dm-3 PQ, 1 mol dm-3 isopropunol, lxl0 -5 mol dm"3 phosphate buffer and NaCIO4, dose=8.9-9.0 Gy.

// i

0

O

|

0.005 [phos. buffer]/mol dm"]

O.01

at

Fig. 5. Effect of phosphate concentration on the 2ridorder decay rate constant observed 490 tim. Pulse radiolysis of N~O-saturated solutions containing 1.0× 10.4 tool dm"J PQ and 0.5 tool dm"3 isopropanol. pH=7.3i-0.1, dose=8.2 Gy, I=0.11 mol dm-3.

Discussion of the Mechanism of PQ Reduction Since the nature of the final transient species found on the one-electron reduction of PQ can not be unequivocally identified, a definite mechanism which explains the experimental observations can not been given. One explanation is a fast protonation process as in the case of some beterocyclic radical anions in aqueous solutions. However, this explanation is in disagreement with the salt-effect experiments on the decay of the 490 nm and 510 nm species. In addition, if the 365 nm absorption decayed by protonation, one would expect the decay to be accelea'ated by phosphate buffer. It was found however that the decay rate constant showed no change with change in the buffer concentration from 0 to lxl0 -2 mol dm-3. Therefore the mechanism described above may be ruled out.Another possibility which would explain the observations in the eaq" reaction with PQ involves tautomerism between the phenanthroline-like radical (an electron resides on one of the pyridine rings) and the semiquinone radical ( an electron resides on one of the oxygen atoms) (Fig. 6). At pH >7, the initial product

548

9th International Meeting on Radiation Processing

formed at the end of the pulse may be a phenanthroline-like radical anion (I)which may undergo a rearrangement to form the PQ serniquinone (PQ*-). This is supported by the reduction potential data (Krishnsn et al. 1983; McWhirter et al., 1990) that phenanthroline free radicals are strong reducing agents and hence are not as stable as their semiquinone counterparts. Both of the radical ion species exist, in principle, in acid-base equilibrium with their respective neutral forms, (]/) ~nd 0II). It may also be that an OH- adds to PQ*" to give a dianion radical, (PQOI-I)*2". On this basis, the dianion would decay by a second-order process to give the product(s) which contribute(s) to the residual absorption at 490 nm on millisecond time scale. At pH--4, the phenanthroline-like radical anion is protonated quickly so the first species observed at the end of the pulse is a N-protonated radical (11)in which the radical center is at the carbon atom adjacent to the protonated nitrogen as in the case of the monoprotonated radical of 1,10-phenanthroline (Janovsky et al., 1978). The rearrangement of (I/) produces an O-protonated PQ semiquinone radical (PQH*)(n[) which may decay via bimolecular disproportion: 2PQH* ~ PQH2 + PQ (2)

(I)

(Pq"l

tt (II) Fig. 6.

(III)

A possible mechanism for the reaction of PQ with eaq-.

REFERENCES

Bao H., Navaratuam S., Parsons B. J. and Phillips G. O. (1992). Pulse radiolysis of phenanthrolinequinoneand iron-PQ complexes. International Conference on "The Biochemistry of Free Radical Formation and Scavenging", Jan. 6th8th, Manchester. Bao H., Navaratuam S., Parsons B. J. and Phillips G. O. (1993). One-electron oxidation of the iron (11)complex of 1,10phenanthroline-5,6-quinone. Radiation Physics and Chemistry, ~ 989. Bao H., (1992). Pulse radiolysis of phenanthrenequinoneand phenanthrolinequinoneand its iron complexes. Ph.D. thesis. University of Salford, UK. Dickeson J. E and Summers L. H. (1970). Derivatives of 1,10-phenanthroline-5,6-quinone. Aust. J. Chem., 23, 1023. Fielden E. M. (1982). Chemical dosimetry of pulsed electron and z-ray sources in the 1-20 MeV range. In: The Study of Fast Processes and Transient Species byElectron Pulse Radiolysis (Baxendale J. H. and Bnsi F., eds), pp. 49-52, D. Reidel Pus. Co., London. Goss C. A. and Abruna H. D., (1985). Spectral, electrochemical, and eleetrocatalytic properties Of 1,10-phenanthroline-5,6dione complexes of transition metals. Inorg. Chem., 24, 4263. Janovsky I. and Teply J. (1978). Pulse radiolysis of aqueous 1,10-phenanthroline solutions. Radiochem. Radioanal. Lett., 34, 361. Koft E. and Case F. H. (1962). Substituted 1,10-phenanthrolines, XIL Benzo and pyrido derivatives. J. Org. Chem., 27, 865. Krishnan C. V., Crentz C., Schwarz H. A. and Sutin N. ( 1983). Reduction potentials for 2,2'-bipyrodine and 1,10phenanthroline couples in aqueous solutions. J. Am. Chem. Sot., 105, 5617. McWhirter R. B. and K.lapper M. H. (! 990). Semiquinone radicals ofmethylamine dehydrogenase, methoxatin, and related o-quinones: A pulse radiolysis study. Biochemistry, 29, 6919. Navaratuam S., Bao H., Parsons B. J. and Phillips G. O. (1989). Pulse radiolysis of some phenanthrolinequinones. Meeting of Fast Reactions and Reactive Intermediates Symposium. Dee., University of York, UK. Smith G. F. and Cargle Jr. F. Win. (1947). The improved synthesis of 5-nitro-l,10-phenanthroline.J. Org. Chem., 12,781. Van der Meer R. A., Jongejan J. A. and Duine J. A. (1988). Pyrroloquinoline quinone is the organic cofactor in soybean lipoxygenase-1. FEBS Lett., ~ 194.