J . Am. Chem. SOC.1991, 113, 3146-3153
3146
Chemistry of Peroxidic Tetrahedral Intermediates of Flavin Giibor Merinyi* and Johan Lind Contribution from the Departments of Physical Chemistry and of Nuclear Chemistry, Royal Institute of Technology, S-10044 Stockholm, Sweden. Received June 13, 1990. Revised Manuscript Received November 26, I990 Abstract: By means of pulse radiolysis 4a-peroxy intermediates of normal and 5-alkylated flavins were produced and the kinetics of their decay into flavin and the corresponding hydroperoxide was investigated as a function of the pH. The neutral and proton-catalyzed breakdown of the 4a-intermediates of 5-alkylated flavinium cations on the one hand and of 5-protio flavins on the other was very similar. It was concluded that the rate-determining step in the neutral decomposition of normal flavin 4a-peroxidesis a heterolysis along the C(4a)-O bond which is catalyzed by water as a general acid. The species initially produced consist of a N(5)-protonated flavinium cation, a neutral hydroperoxide, and a hydroxide ion. The process is completed by rapid deprotonation of the flavinium cation to yield the neutral flavin. By combination of kinetic and thermodynamic data determined in this and other laboratories, the energetics of the autoxidation of 1 ,Sdihydroflavin was resolved into individual steps. The proton-catalyzed breakdown of flavin 4a-peroxides is initiated by a proton-assisted expulsion of neutral hydroperoxide leaving behind the N(5)-protonated flavinium cation. The attenuation of proton catalysis with decreasing pH indicates thermodynamic protonation of the 4a-intermediates around pH 3. The site of protonation is presumably the N(5) or the N( IO) atom. The hydroxide ion catalyzed breakdown of the 4a-species is best interpreted by assuming the rate-determining step to be deprotonation of the N(5)-H site followed by rapid expulsion of the hydroperoxide anion and neutral flavin. This picture demands the microscopic pK, of the N(5)-H group to be below 17. The possible role of enzymes in stabilizing the 4a-intermediates against breakdown into flavin and hydroperoxide is discussed. It is suggested that an apolar, hydrophobic pocket may be the chief stabilizing factor. In such an environment, the transition state for heterolysis and homolysis may approach each other. Finally, the bond strength of the peroxidic 0-0 bond was calculated from recent thermodynamic data. This bond turns out to be weaker (e26 kcal/mol) than the 04bond in any known linear peroxide. From the finding that the 04bond is weaker than the C(4a)-O bond it is argued that, in sufficiently hydrophobic enzymes, monooxygenation may be initiated by homolysis of the 0-0 bond. It is suggested that the comparable strengths of the C(4a)-0 and 0-0bonds may be the prime reason for the versatility of flavin enzymes.
Introduction Many vital functions of flavin enzymes involve the transient presence of intermediates characterized by a C(4a)-0 bond. For definitions, see formula FIRSOOR. The most important inter-
k FIRSOOR
mediate is a flavin C(4a)-hydroperoxide, which operates in monooxygenases and hydr0xy1ases.l~~Bound to the Vibrio Harveyi luciferase enzyme, this species has been positively identified by I3C N M R ~pectroscopy.~Bacterial luminescence is generally believed to arise from the decomposition of a flavin C(4a)-pero~yhemiacetal~-~ bound to a luciferase enzyme. Although strongly implicated by kinetic e~idence,~,’ this species has never been isolated or even characterized unequivocally. The decomposition of the peroxyhemiacetal is believed to produce the enzyme-bound pseudobase7~*in an excited state from whence chemiluminescence ( I ) Massey, V.; Hemmerich, P. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1975; Vol. XII, pp 191-252. (2) Ballou, D. P. In Flavins and Flauoproteins; Bray, R.C., Engel, P. C., Mayhew, S. G., Eds.; de Gruyter: Berlin, 1984; pp 605-618. (3) Vervoort, J.; Miiller, F.; Lee, J.; van den Berg, W. A. M.; Moonen, Ch. T. W. Biochemistry 1986, 25, 8062. (4) Eberhard, A,; Hastings, J. W. Biochem. Biophys. Res. Commun. 1972, 47, 348. (5) Kurfiirst, M.; Ghisla, S.; Hastings, J. W. Proc. Natl. Acad. Sci. U.S.A. 1984,81, 2990. (6) Macheroux, P.; Ghisla, S.;Kurfiirst, M.; Hastings, J. W. In Flauins and Flavoproteins; Bray, R.C., Engel, P. C., Mayhew, S.G., Eds.; de Gruyter: Berlin, 1984; pp 669-672. (7) Ghisla, S.; Eckstein, J.; Macheroux, P. In Flavins and Flauoproteins; Edmondson, D. E., McCormick, D. B., Eds.; de Gruyter: Berlin, 1987; pp 601-612.
0002-7863/91/1513-3146$02.50/0
occurs. The isolation of the pseudobase and the finding that its fluorescence (albeit weak) is superimposable on the bioluminescence spectrum lends strong support to this vie^.^,^ In many monooxygenase enzymes the bound 4a-hydroxide has been observed* (judging from spectral properties) prior to its reverting to flavin and water. With an eye to understanding the role of flavin enzymes, much effort has been expended in the past to investigate the chemistry of model C(4a)-hydropero~ides.~*~~ These studies necessitated alkylation of the N(5) position of the flavin, a process that apparently entails thermodynamic stabilization of the tetrahedral species in relation to the parent flavinium cation. What is still lacking is a comparative study to pinpoint the factors that affect the breakdown kinetics of the peroxidic species. The present work encompasses the kinetic investigation of several flavin C(4a)peroxy species as a function of site alkylation and the pH of the water solvent. Results C(4a)-hydroperoxidesof N(5)-H Flavins. In a series of papers has demonstrated the facile generation, by means of pulse radiolysis, of C(4a)-hydroperoxides of normal flavins (Fl), such as riboflavin and flavin mononucleotide (FMN). Although these species could not be positively identified, spectral comparison with enzyme-bound C(4a)-hydroperoxides and 5-alkylated model (8) Kurfiirst, M.; Hastings, J. W.; Ghisla, S.; Macheroux, P. In Flavins and Flavoproteins;Bray, R. C., Engel, P. C., Mayhew, S. G., Eds.; de Gruyter: Berlin, 1984; pp 658-667. (9) Lee, J. In Chemi- and Bioluminescence; Burr, J. G., Ed.; Marcel Dekker: New York, 1985; pp 401-437. (10) Kemal, C.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1976, 73,995. ( 1 1) Kemal, C.; Chan, T. W.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 405. (12) Bruice, T. C. In Flavins and Flauoproteins; Bray, R.C., Engel, P. C., Mayhew, S. G., Eds.;de Gruyter: Berlin, 1984; pp 46-55, and references cited
therein. ( 1 3) Bruice, T. C. Isr. J. Chem. 1984, 24, 54, and references cited therein. (14) Keum, S.-R.;Gregory, D. H.; Bruice, T. C. J . Am. Chem. Soc. 1990, 112, 2711. ( 1 5) Anderson, R. F. In Oxygen and Oxy-radicals in Chemistry and Biology; Rodgers, M. A. J., Powers, E. L., Eds.; Academic Press: New York, 1981; pp 597-600. (16) Anderson, R.F. In Flauins and Flauoproteins; Massey, V., Williams, C. H., Eds.; Elsevier: Amsterdam, 1982; pp 278-283.
0 1991 American Chemical Societv
J . Am. Chem. SOC.,Vol. 113, No. 8,1991 3147
Peroxidic Tetrahedral Intermediates of Flavin
1
Table 1. Yields per Pulse of Flavin C(4a)-PeroxidesRelative to their Maximum Yields as a Function of the pH" PH C(4a)-OOR DH C(4a)-OOH
86
5.0 5.5 5.8 6.0
100
5.9
IO0
100
a
0
99
6.4
88
6.8 7. I 7.7 7.9 8.1
88 76 71 65
5 n
61
7.4 7.7
67 47
7.8 8.0 8.3
41 35
8.4 44 28 9.1 39 9.1 17 9.3 33 9.3 e10 'The dose per pulse was 200 Gy. R denotes (CH3)2C(OH)CH2. C(4a)-hydroperoxides, as well as a similarity in chemical behavior in several enzymes of authentic C(4a)-hydroperoxide and radiolytically generated intermediate,17 leaves little doubt as to the nature of these species. A brief description of the experimental technique is appropriate. Irradiation of an oxygenated aqueous formate solution rapidly generates CO2*- and 02*-. The former reduces FI to F1'- and O2 to 02'-, and these radical anions protonate according to their pK,'s. By tailoring the system a desired ratio FIH*/O2'- can be obtained. The hydroperoxide forms according to reactions 1-3 with k , = IO9, k2 = 5 X lo8, and k3 = FIH' F1H'
+ HO2'
FIHO2H
2FIH' FI + FIH2 (4) promote reactions 1-3 at the expense of 4, 02'-was produced in a 5-10-fold e x m over FIH'. With aerated solutions, this required the FI concentration to be well below M. Apart from the competition of reaction 4 with 1-3, which is independent of the radical concentration generated in a single pulse, i.e. the dose, there are additional reactions, to be mentioned below, that may further reduce the yield of FIH02H. (a) At very high doses the initial reductant, C02'-, can reduce the semiquinone radical, FIH', to fully reduced flavin in reaction 5.
+ FIH'(FI'-)
HP
C02 + FIH,(FIH-)
(5)
(b) When the pH is raised above ca.7.5, the FlH' radical with a pK, of ca.8.31e21 starts to dissociate into FP-.'lF reacts rapidly with O2 in competition with its reaction with 02'-. F1'-
+0 2
-+
0.01
0.02
time (s)
-2
I:\
FlHO2H
-OH-
2 X IO8 M-' s-I. At 460 nm, where FI absorbs but FIH02H is almost transparent, the kinetics of the breakdown of FlH02H into FI and H 2 0 2has been m e a s ~ r e d ' ~in . l ~the pH interval 5-8. Using a similar technique, we remeasured these rates as well as the corresponding rates with 3-methylated tetraacetylriboflavin (3Me-TARF), extending the pH range from pH 1 to ca. IO. It will be recalledI8 that FIH' undergoes the dismutation reaction 4. In order to
C02'-
0
(1)
-
+ 02'-
-+
H2 0
-0.25
FI
+ 02'-
k3 and k6 have comparable magnitudes ((2-3)
X IO8
(6) M-l s-I 122,23
(17) Massey, V.; Schopfer, L. M.; Anderson, R . F. In Oxidases and Reluted Redox Systems; King, T. E., Mason, H. s.,Morrison, M. M., Eds.; Alan Liss: New York, 1988; pp 147-166. (18) Lind, J.; MerCnyi, G. Phofochem. Photobiol. 1990, 51, 21. (19) Ehrenberg, A.; Muller, F.; Hemmerich, P. Eur. J . Biochem. 1967. 2, 286. (20) Draper, R. D.; Ingraham, L. L.Arch. Biochem. Eiophys. 1968, 125, 802. (21) Land, E. J.; Swallow, A. J. Biochemisfry 1969, 8, 21 17.
-5
1
0
0.01
0.02
time (s) Figure 1. Kinetic traces representing the change of absorbance at 460 nm with time. The upper curves show the measured signal while the lower ones represent the same data as log linear plots. In all cases the solutions contain 5 X M riboflavin, 2.6 X lo4 M 02.and 1.2 X M HC02Na. Traces: (a) pH 7.73, initial radical concentration 7 X IO" M; (b) pH 7.73, initial radical concentration -2 X IO4 M; (c) pH 9.05, initial radical concentration -2 X IO-' M.
and therefore a substantial portion of F1'- reverts to FI instead of forming F1H02H. Indeed, in order for reaction 3 to compete efficiently with reaction 6, the 02'-concentration must equal or exceed that of oxygen. Consequently, very high doses are needed, which, however, also brings reaction 5 into play. Obviously, the yield of the hydroperoxide per pulse will always be smaller at high than at low pH. Thus, the signal at 460 nm will be reduced whence the rate measurements become more difficult. Table I shows the initial yield of F1HO2H as a function of the pH at a dose of 200 Gy/pulse, which produces a total radical concentration of ca. 1.2 X M. From the above points a and b it transpires that, generally, the bleaching observed at 460 nm by ca. 2 X lo4 s after the pulse (the time during which all semiquinone radicals, FIH' (FI*-) have essentially disappeared) is due to the presence of FIH02H and FIH2(FIH-). The rate of reformation of fully oxidized flavin should then be given by the sum of two exponentials. Figure 1 presents kinetic traces that demonstrate the features to be described forthwith. Below pH 7.5 the kinetic traces fit to a single exponential irrespective of the dose, flavin, and 0, concentration. This is not shown in the figure as the low-pH traces are similar to trace a. Above pH 7.5 a biphasic process is observed at high doses (see trace b in Figure 1). The rate of the rapid (22) Vaish, S.P.; Tollin, G.Bioenergefics 1971, 2, 61. (23) Faraggi, M.; Hemmerich, P.; Pecht, 1. FEBS Lett. 1975, 51, 47.
Merenyi and Lind
3148 J . Am. Chem. SOC.,Vol. 113, No. 8, 1991 Table 11. Rate Constants of Decomposition of Flavin C(4a)-O-X species into Fully Oxidized Flavin and XOH‘ e10
R5
X
R’
H OH OH OH OH XIC
CH3 H CH’ H
C2H5
RlO TARO
H H
ribose TAR
kH
0 1
s-3
0.01 300 36
kH1,
M-1
3 x 10’ 106
5 x 105 1.1 x 105 I x 105
kOH,
kH29
M-I
s-I
M-1
s-I
3 x 102 4 x 104 6 X IO’
nm RPb 21 TAR 28 1.6 x 103 CH’ H ribose nm H 820 106 nm X2d H H ribose 520 nm “Tetraacetyl ribose. Ribose 5’-phosphate. CCH3CH(OH)0.d(CH3)2C(OH)CH,0. cnm, no measured. ClH5 CzH5
s-l
kH20/ kD20
5 x 108 5 x 107 no
3.0 & 0.2 2.5 0.2 nm 2.2 f 0.2 nm nm
*
no
3 x 108 108
ligible. For the 5-alkylated species, KOH is set equal to zero (see below).
10‘ I
FIH200H+ s FIHOOH
+ H+
(8)
The dotted line is computed with eq 11, i.e., by neglecting the kobs
io’ 1 0
2
6
4
8
i
10
PH Measured rates of breakdown into flavin and hydrogen peroxide of three flavin 4a-00H species in water as a function of the pH. V, FIH-4a-OOH: 0 , 3MeTARFH-4a-OOH; 5TARF-4a-OOH. Figure 2.
reformation of FI increases with increasing pH while the rate of the slow component decreases in the pH 7.5-9 range, above which it assumes a pH-independent value (cf. traces b and c in Figure I ) . By varying the dose in the pH range 7.5 < pH < 10 it was possible to extract the rate constant of reaction 7 from the rapid FIHOOH
---L
FI
+ H202
(7)
component of the kinetic trace. Above pH IO the rate of formation of FIHOOH is lower than that of reaction 7, making measurements impossible. Figure 2 presents the measured rate of reaction 7 as a function of the pH for three flavin C(4a)-hydroperoxides. Apart from a small factor, the rates of the hydroperoxides of normal and 3methylflavin show parallel pH dependences in the whole pH regime. This holds equally for the hydroperoxide derived from the 3-methyl-5-ethyltetracetylriboflaviniumcation (SEt-TARF+) at pH
