P h o r o c h r a i w y wid Phvrohiolo!i?. 1975, Vol 21. pp 205-208.
Pcrgamon Press
Printed in Great Britain
RESEARCH NOTE FLAVIN-SENSITIZED PHOTOOXIDATION OF HISTIDINE* PAULG. JOHNSON,ANNE P. BELL? and DONALD €3. MCCORMICKS Section of Biochemistry, Molecular and Cell Biology, and Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, U S A .
(Received 12 July 1974; accepted 4 Nooember 1974)
Flavins are known to sensitize the aerobic photooxidations of amino acids (Galston, 1950; Frisell et al., 1959). For aromatic amino acids, there is some evidence that triplet flavin produces singlet oxygen, which reacts with the aromatic ring (Penzer, 1970). In particular, Tomita et al. (1969), who studied the flavinsensitized photooxidation of histidine, demonstrated that a 1,Ccycloadditionof singlet oxygen to the imidazole ring produced a cyclic peroxide, which decomposed to give aspartic acid via at least 17 intermediates. This photosensitized decomposition of amino acids by flavin can be used to induce a chemical modification of proteins (Penzer, 1970; Taylor and Radda, 1971). More importantly, the naturally bound coenzyme in a flavoprotein can act as a photosensitizer, causing reaction of nearby amino acid residues and thus helping to elucidate the environment of the binding site (McCormick, 1970;Tu and McCormick, 1973). Recently, several flavin-containing enzymes have been shown to have a histidine residue at the active site. Succinate dehydrogenase (Walker and Singer, 1970), D-6-hydroxynicotine oxidase (Mohler et al., 1972),and sarcosine dehydrogenase (Patek and Frisell, 1972) actually have the N-3 nitrogen of the imidazole ring of the histidine moiety covalently attached to the 8a-position ofa FAD. An absorption spectral study on glucose oxidase (Miiller et al., 1970)allows for the presence of a histidine near the FAD-binding site, but, in this case, the imidazole ring is not covalently attached to the flavin. These developments prompted us to study the ability of the light-excited flavin to oxidize histidine, as was done previously for other aromatic amino acids (Wu and McCormick, 1971). Flavinyl histidines, pre~
*This work was supported in part by Research Grant AM-04585 from the National Institute of Arthritis, Metabolism. and Digestive Diseases, U S . Public Health Service, and in part by funds made available through the State University of New York. tPredoctoral trainee supported by Ford Minority Fellowship to the Section of Biochemistry, Molecular and Cell Biology. $Person to whom all correspondence and reprint requests should be addressed.
viously synthesized to study imidazole-isoalloxazine interactions (Johnson and McCormick, 1973), were used as models for this work. These compounds simulate the close association that occurs in flavoproteins and also have the nucleophilic amino group of the amino acid tied up as an amide bond. The structures of these compounds and the parent flavin acids are shown below. COOH
I
cH3axy0 (CH2)2,4.5
I
CHI
NH
0
12.4.3
0
H
CHp
I1 I
I
I
C -N-CH
-COOH
(CHi?)Z ,5
cH3 C H I ~y--~O
0 p2,3,4,5
MATERIALS AND METHODS
The 10-w-carboxyalkylflavins (12-5) were synthesized according to the method of Fory et al. (1968). The flavinyl histidines (112-5) were prepared as described by Johnson and McCormick (1973). FMN (riboflavin 5’-phosphate), FAD (flavin-adenine dinucleotide), L-histidine, and N-acetyl-L-histidine were purchased from Sigma. Histidine methyl ester dihydrochloride was synthesized by the method of Fischer and Cone (1908). p-Nitroaniline was obtained from Aldrich. The flavins were activated at 445 nm using the 150 W xenon lamp of an Aminco-Bowman spectrophotofluorometer. The cell compartment was thermostated at either 25,35 or 50°C with a Haake Model F constant temperature circulator. The flavin concentrations were determined from the absorbance at 450 nm, at which wavelength they have a molar extinction coefficient of approximately 12 x lo3.All work was performed in a dark room. The aerobic photochemistry was performed in open 4-mY quartz cuvettes with 1 cm pathlengths. There was 1 slit
205
206
P. G . JOHNSON, A. P. BELLand D. B. MCCORMICK
(3mm) between the light source and cuvette. The stirred M in 0.05 M sodium phosphate or acesolutions (3 x tate buffer) contained either flavinyl histidine or equimolar solutions of L-histidine (or its derivative) and flavin or flavinyl histidine. The ability of the flavin to sensitize photooxidations was measured by removing aliquots (0.3 m y )from the reaction mixture at various times and determining the remaining histidine coiorimetrically, using a modification of the Pauly reaction. The assay used consisted of coupling diazotized p nitroaniline with the imidazole ring of histidine and measuring the color at 550 n m as described by Tabor (1957). The anaerobic photochemistry was performed on 8 x lo-' M solutions (in 005 M buffer) in 4-my Thunberg cuvettes ( I cm pathlength). The cuvettes were evacuated with a water aspirator and refilled with nitrogen gas. This 40c process was repeated several times over a period of 20 min. The nitrogen gas was made free of oxygen by passing it 0 I 2 3 through 3 scrubbers containing vanadous sulfate, sulfuric Hours o f Illumination acid, and amalgamated zinc. The anaerobic cuvettes were illuminated(no slits)at 25°C in the same manner as the aer- Figure 1. Flavin-sensitized photooxidations of histidine obic ones. The extent of flavin photoreduction was mea- and derivatives at pH 8. Aqueous solutions contained 3 x sured by the decrease in the 445-nm absorbance, using a M flavinyl histidine (112,5)or flavin and histidine in Beckman DU spectrophotometer. aerobic 0.05 M sodium phosphate bufler (25°C). Fluoresence polarization measurements were made on the Aminco-Bowman spectrophotofluorometerwith 3-mm slits, lP2l photomultiplier tube, and Clan-Thompson attachments. Flavin M ) , in 0.01 M sodium phosphate rates for decomposition of the free bases were slightly buffer at pH 7.5. was diluted with glycerol-water solutions greater than that of the Ncr-substituted analogues. to make 2 x 10- M flavin in 2 x 10- M buffer, with glyA previous study (Johnson and McCormick. 1973), cerol varying from 59-84 per cent by weight. The procedure indicating that the imidazole ring of the flavinyl histiand calculations were as described by MacKenzie er al. dines is partially protonated at pH 7. prompted the (1969). investigation of flavin-sensitized photooxidation of histidine at pH 8 and 2S°C, as shown in Fig. 1. As RESULTS AND DISCUSSION expected, the rates of oxidation of histidine by interArrobic photochemistry. The rates of flavin-sensit- molecularly added 10-w-carboxyalkylflavins (J2 and ized photooxidations of histidine and its derivatives 14) are only slightly greater at pH 8.0 than at pH 7.0. were determined by measuring the disappearance of The rates of histidine decomposition in the case of the the imidazole residue. This was done by diazotizing the flavinyl histidine amides (112 and 11,) are much slower histidine with Pauly's reagent and estimating the loss than the intermolecular cases with I,, I,, and 1,. This of color at 550 nm. The essence of this assay is that the curtailment in rate for the amide cases may, in part, rereagent must be much more sensitive to the histidine flect intramolecular, ground-state complex formation moiety than to the oxidation products. This assump- between flavin and histidine. Such complexing detion was justified by previous workers (Tomita et al., creases the fraction of flavin in excited forms capable 1969),who studied the photooxidations of N-benzoyl- of initiating the photooxidation of the histidine moiety. histidine and histidine as sensitized by rose bengal, A fluorescence and PMR study (Johnson and McCormethylene blue, and riboflavin. mick, 1973), in which the quenching of the 520-nm flaThe ability of several flavins to photosensitize inter- vin fluorescence emission and the chemical shifts of the molecularly the oxidation of histidine and its deriva- imidazole and isoalloxazine ring protons were studied tives at pH 7 and 25°C was measured. The rates of at various pH values and temperatures, definitely reactions for F M N and the 10-w-carboxyalkyltlavins showed some nonfluorescent, ground-state complex with histidine are similar, but F M N is 4 times more formation. Furthermore, this particular type of intereffective a sensitizer than FAD. This diminished pho- action is greater for the short-chain flavinyl histidine toreactivity of FAD can be attributed to intramolecu- (11,) than for the longer-chain one (IJs). Examination lar complex formation between the adenine and flavin of the photooxidation data in Fig. 1 further substanmoieties (Wu and McCormick, 1971; Weber, 1950; tiates the conclusions drawn from the fluoresccnce and McCormick et al., 1967). The flavin acid (I,) sensitizes PMR data, i.e. the rate of decomposition of the shortthe photooxidation of the histidine derivatives, i.e. chain amide (Ilz) is slower than the long-chain one N-acetyl-L-histidine and histidine methyl ester dihydro- (I15). Although not shown graphically, the rates of chloride, to a slightly greater degree than it does the photooxidation of amides 11, and 11, lie between free amino acid. After 3 h of illumination, 53 per cent those of 11, and 11,. The rates are in the following of the histidine remains, compared to only 44 per cent decreasing order: 115 > 11, z 11, > [Iz. of the N-acetyl derivative. This differs from results The extent of ground-state complex formation can obtained for the intermolecular FMN-sensitization of be semiquantitatively estimated using fluorescence tryptophan, tyrosine, and their N-acetyl derivatives polarization. The double-reciprocal plot of polariza(Wu and McCormick, 1971). In the latter cases, the tion of fluoresence of an intermediate-length flavinyl
Research Note
201
I
0
2
4
6
8
0
10
1 '7)
1
I I I 3 4 5 Hours of Illumination I
2
6
Figure 2. Double-reciprocal plot of polarization of fluorescence of flavinyl histidine (11,) and free flavin (I,) at pH 7.5 and 22°C in aqueous glycerol solutions of varying viscosity.
Figure 3. Anaerobic photolysis of histidine and derivatives at pH 8. Aqueous solutions contained 8 x 10- ' M flavinyl histidine (112,5) or flavin and histidine in 005 M sodium phosphate buffer (25°C).
histidine (11,) and flavin acid (I4) a t pH 7.5 in aqueous glycerol solutions of varying viscosity is shown in Fig. 2. From the ratio of the slopes (1-8), which is directly proportional to the ratio of mean lifetimes, and from the ratio of fluorescence of free flavin to flavin amide, which was determined as 2.8, it can be calculated (MacKenzie et al., 1969) that approximately 80 per cent of the quenching in 11, is due to ground-state complex formation. The simple demonstration of intramolecular complexing for the amide does not unequivocally show that such of the flavin moiety as becomes excited is unable to photooxidize the histidine. It may be that some of the excitation energy is dissipated by collisional interactions. There isalso the possibility that the imidazole and isoalloxazine rings are not as frequently mutually oriented in such a manner as to give proper oxidative interaction. This latter possibility seems unlikely, since the additional decomposition of histidine added to the amides is rather slight. These results tend to support the tenet that ground-state complexing impairs formation of the light-excited flavin, which may additionally be reverted to ground state by nonphotochemically productive collisions. Hence, decrease in the photooxidation of histidine within the amides is not mainly attributable to steric factors. The effect of intermolecularly added flavin on the photooxidation of histidine within flavinyl histidine was also investigated. The rates of decomposition of the mixtures of flavinyl amide and flavin (11, and 1,; 115 and I,) at pH 8 are much greater than for amide alone. For example. after 3 h of illumination, there is twice as much histidine remaining in the case of 11, as compared to the same amide to which equimolar 10-0cdrboxypentylflavin (I5) has been added. Further comparison shows that the rate of photooxidation is greater for the case of histidine and flavin (I,) than for flavinyl histidine (1I2,,) and flavin (12,5J. This suggests that the histidine within the amide is less accessible to added free Havin than that in a mixture of flavin plus histidine. The further importance of stereochemistry
can also be appreciated by the fact that the rate of decomposition of the larger and more bulky amide (115 15) is slower than the smaller one (11, I,). This agrees with similar measurements made on flavinyl tryptophans and tyrosines (Wu and McCormick, 1971). Previous work (Johnson and McCormick, 1973), in which the fluorescence-pH profiles of the flavinyl histidines were studied, showed that a t p H 4 and below there were very few isoalloxazine-imidazole interactions. This prompted the investigation of the rates of flavin-sensitized photooxidations of histidine at pH 4.0. The equimolar mixture of histidine and either FMN or 10-w-carboxybutylflavin (I4) shows a much slower degradation rate at pH 4 than at pH 7 or 8. This result agrees with a previous study (Taylor and Radda, 1971), which indicated very little photooxidation of histidine when the imidazole ring is protonated. The rates of photooxidation of flavinyl histidines (112.5) are lower at pH 4 than a t pH 8. The intermolecular addition of histidine or FMN to the amides has little effect on the rates. Thus, in the case of the flavinyl histidines, not only do the histidine and flavin moieties show very little association at p H 4 (Johnson and McCormick. 1973), but the ability of the flavin to sensitize the photooxidation of the protonated imidazole ring is also markedly decreased. The effect of temperature on photooxidation of the flavin amides follows an optimum. The increase in rate upon elevating the temperature above 25°C is probably due to greater dissociation of the ground-state complex, allowing the 450-nm light to create more triplet flavin. However, as the temperature increases, so do the molecular collisions, which causes quenching of the excited state. This probably explains why there is a decrease in rate when going from 35 to 50°C. Anaerobic photochemistry. The anaerobic oxidation of amino acids to keto acids and ammonia by lightexcited flavin provides a close analogy to amino acid oxidase systems. Byrom and Turnbull (1970) propose that triplet flavin abstracts hydrogens from the amino
+
+
208
A. P. BELLand D. B. MCCORMICK P. G. JOHNSON,
acid substrate, leading to production of dihydroflavin and concomitant loss of the yellow color. On admission of oxygen into the reduced solution, the oxidized flavin quantitatively reappears. The per cent loss of absorbance at 445 nm, when flavin solutions are irradiated anaerobically a t pH 8, is shown in Fig. 3. The rate of such color loss in the case of F M N plus histidine is much faster than for 10-w-carboxybutylflavin (I,) and histidine. In fact, after 6 h of illumination in the absence of oxygen, there is twice as much oxidized flavin in the I, case than in the F’MN one. However, if oxygen is then admitted to the systems, 92 per cent of the color returns to the histidine solution containing 14, while only 78 per cent of the color returns to that containing FMN. These data seem to indicate that a considerable amount of the F M N undergoes a photobleaching reaction. Anaerobic photobleaching includes a series of reactions in which both the excited singlet and triplet states of the flavin (Song and Metzler, 1967) intramolecularly react with its side chain to give various products, including lumiflavin and lumichrome. Thus, upon admission of air to such a system, only partial restoration of the absorbance (445 nm) is observed. Apparently, the greater part of photobleaching in the cuvette with FMN is due to the presence of the 2’-hydroxyl in the ribityl chain (Moore et al., 1963). Some photobleaching occurs in the I,-histidine system. It is known (Yang and McCormick, 1965) that the presence of a hydroxyl group on the 2’-position of the side chain is optimal, but not necessary, for intramolecular reaction with the isoalloxazine ring.
The anaerobic photolysis of flavinyl histidine amides (II2 and 115) produces less than 5 per cent loss in color, even after 6 h of irradiation at pH 8 and 25°C. This is to be expected, since the reactive amino groups necessary for ammonia production exist as amides in these particular compounds. Although not shown graphically, 112 and 11, retained greater than 90 per cent of their absorbance at 445 nrn after 24 h of constant illumination under anaerobic conditions (pH 8). Upon addition of oxygen, the absorbance returned to only 92 per cent of the original in both cases. As measured quantitatively using Pauly’s reagent, there was no apparent destruction of the imidazole moiety for either 112 or 115. This resistance to photobleaching, and the fact that the short-chain amide reacts slower than the long-chain one, seems again to suggest intrarnolecular, ground-state complex formation between the isoalloxazine and imidazole moieties. This interaction decreases formation of excited-state flavin necessary for initiation of photobleaching reactions. The effect of intermolecularly added L-histidine on the photoreduction and photobleaching of flavin in 112 and 115 is also depicted in Fig. 3. The rates are slightly higher than for the flavinyl amides alone. Admission of air to the IIs plus histidine system after 6 h of illumination returns the color to 99 per cent of its original value. These data seem to indicate that photoreduction, rather than photobleaching, is operating in these cases, and the lower rate is again due mostly to complexation of isoalloxazine and imidazole.
REFER EN CES
B y r o n P. and J. H. Turnbull (1968) Photochem. Photobiol. 8, 243254. Fischer, E. and L. H. Cone (1908) Ann. Chem. 363, 107-117. Fory, W., R. E. MacKenzie and D. B. McCormick (1968) J . Heterocy. Chem. 5,625-630. Frisell, W. R., C. W. Chung and C. G. MacKenzie (1959) J . Biol. Chem. 234, 1297-1302. Galston, A. W. (1950) Science 111,619-624. Johnson, P. G. and D. B. McCocmick (1973) Biochemistry 12,3359-3364. MacKenzie, R. E., W. Fory and D. B. McCormick (1969) Biochemistry 8, 1839-1844. McCormick, D. B. (1970) Experientia 26, 243-244. McCormick, D. B., J. F. Koster and C. Veeger (1967) Eur. J. Biochem. 2,387-391. Mohler, H., M. Briihmiiller and K. Decker (1972) Eur. J. Biochem. 29, 152-155. Moore, W. M., J. T. Spence, F. A. Raymond and S. D. Colson (1963) J . Am. Chem. SOC.85, 3367-3372. MWler, F., P. Hemmerich, A. Ehrenberg, G. Palmer and V. Massey (1970) Eur. J. Biochem. 14, 185-196. Patek, D. R. and W. R. Frisell (1972) Arch. Biochem. Biophys. 150,347-354. Penzer, G . R. (1970) Biochem. J. 116,733-743. Song, P A and D. E. Metzler (1967) Photochem. Photobiol. 6,691-709. Tabor, H. (1957) In Merhods in Enzymology(Edited by S. P. Colowick and N. 0. Kaplan), Vol. 3, pp. 63@-631. Academic Press, New York. Taylor, M.B. and G. K. Radda (1971) In Methods in Enzymology (Edited by D. B. McCormick and L. D. Wright), Vol. 18(B), p. 496. Academic Press, New York. Tomita, M., M. Irie and T. Ukita (1969) Biochemistry 8, 5149-5160. Tu,S.-C. and D. B. McCormick (1973) J . B i d . Chem. 248,633%6347. Walker, W. H. and T. P. Singer (1970) J. Biol. Chem. 245, 42244225. Weber, G. (1950) Biochem. J . 47, 114-122. Wu, F. Y.-H. and D. B. McCormick (1971) Biochim. Biophys. Acra 236,479-486. Yang, C. S.and D. B. McCormick (1965) J. Am. Chem. SOC.87,57635765.
Pcrgamon Press
Printed in Great Britain
RESEARCH NOTE FLAVIN-SENSITIZED PHOTOOXIDATION OF HISTIDINE* PAULG. JOHNSON,ANNE P. BELL? and DONALD €3. MCCORMICKS Section of Biochemistry, Molecular and Cell Biology, and Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, U S A .
(Received 12 July 1974; accepted 4 Nooember 1974)
Flavins are known to sensitize the aerobic photooxidations of amino acids (Galston, 1950; Frisell et al., 1959). For aromatic amino acids, there is some evidence that triplet flavin produces singlet oxygen, which reacts with the aromatic ring (Penzer, 1970). In particular, Tomita et al. (1969), who studied the flavinsensitized photooxidation of histidine, demonstrated that a 1,Ccycloadditionof singlet oxygen to the imidazole ring produced a cyclic peroxide, which decomposed to give aspartic acid via at least 17 intermediates. This photosensitized decomposition of amino acids by flavin can be used to induce a chemical modification of proteins (Penzer, 1970; Taylor and Radda, 1971). More importantly, the naturally bound coenzyme in a flavoprotein can act as a photosensitizer, causing reaction of nearby amino acid residues and thus helping to elucidate the environment of the binding site (McCormick, 1970;Tu and McCormick, 1973). Recently, several flavin-containing enzymes have been shown to have a histidine residue at the active site. Succinate dehydrogenase (Walker and Singer, 1970), D-6-hydroxynicotine oxidase (Mohler et al., 1972),and sarcosine dehydrogenase (Patek and Frisell, 1972) actually have the N-3 nitrogen of the imidazole ring of the histidine moiety covalently attached to the 8a-position ofa FAD. An absorption spectral study on glucose oxidase (Miiller et al., 1970)allows for the presence of a histidine near the FAD-binding site, but, in this case, the imidazole ring is not covalently attached to the flavin. These developments prompted us to study the ability of the light-excited flavin to oxidize histidine, as was done previously for other aromatic amino acids (Wu and McCormick, 1971). Flavinyl histidines, pre~
*This work was supported in part by Research Grant AM-04585 from the National Institute of Arthritis, Metabolism. and Digestive Diseases, U S . Public Health Service, and in part by funds made available through the State University of New York. tPredoctoral trainee supported by Ford Minority Fellowship to the Section of Biochemistry, Molecular and Cell Biology. $Person to whom all correspondence and reprint requests should be addressed.
viously synthesized to study imidazole-isoalloxazine interactions (Johnson and McCormick, 1973), were used as models for this work. These compounds simulate the close association that occurs in flavoproteins and also have the nucleophilic amino group of the amino acid tied up as an amide bond. The structures of these compounds and the parent flavin acids are shown below. COOH
I
cH3axy0 (CH2)2,4.5
I
CHI
NH
0
12.4.3
0
H
CHp
I1 I
I
I
C -N-CH
-COOH
(CHi?)Z ,5
cH3 C H I ~y--~O
0 p2,3,4,5
MATERIALS AND METHODS
The 10-w-carboxyalkylflavins (12-5) were synthesized according to the method of Fory et al. (1968). The flavinyl histidines (112-5) were prepared as described by Johnson and McCormick (1973). FMN (riboflavin 5’-phosphate), FAD (flavin-adenine dinucleotide), L-histidine, and N-acetyl-L-histidine were purchased from Sigma. Histidine methyl ester dihydrochloride was synthesized by the method of Fischer and Cone (1908). p-Nitroaniline was obtained from Aldrich. The flavins were activated at 445 nm using the 150 W xenon lamp of an Aminco-Bowman spectrophotofluorometer. The cell compartment was thermostated at either 25,35 or 50°C with a Haake Model F constant temperature circulator. The flavin concentrations were determined from the absorbance at 450 nm, at which wavelength they have a molar extinction coefficient of approximately 12 x lo3.All work was performed in a dark room. The aerobic photochemistry was performed in open 4-mY quartz cuvettes with 1 cm pathlengths. There was 1 slit
205
206
P. G . JOHNSON, A. P. BELLand D. B. MCCORMICK
(3mm) between the light source and cuvette. The stirred M in 0.05 M sodium phosphate or acesolutions (3 x tate buffer) contained either flavinyl histidine or equimolar solutions of L-histidine (or its derivative) and flavin or flavinyl histidine. The ability of the flavin to sensitize photooxidations was measured by removing aliquots (0.3 m y )from the reaction mixture at various times and determining the remaining histidine coiorimetrically, using a modification of the Pauly reaction. The assay used consisted of coupling diazotized p nitroaniline with the imidazole ring of histidine and measuring the color at 550 n m as described by Tabor (1957). The anaerobic photochemistry was performed on 8 x lo-' M solutions (in 005 M buffer) in 4-my Thunberg cuvettes ( I cm pathlength). The cuvettes were evacuated with a water aspirator and refilled with nitrogen gas. This 40c process was repeated several times over a period of 20 min. The nitrogen gas was made free of oxygen by passing it 0 I 2 3 through 3 scrubbers containing vanadous sulfate, sulfuric Hours o f Illumination acid, and amalgamated zinc. The anaerobic cuvettes were illuminated(no slits)at 25°C in the same manner as the aer- Figure 1. Flavin-sensitized photooxidations of histidine obic ones. The extent of flavin photoreduction was mea- and derivatives at pH 8. Aqueous solutions contained 3 x sured by the decrease in the 445-nm absorbance, using a M flavinyl histidine (112,5)or flavin and histidine in Beckman DU spectrophotometer. aerobic 0.05 M sodium phosphate bufler (25°C). Fluoresence polarization measurements were made on the Aminco-Bowman spectrophotofluorometerwith 3-mm slits, lP2l photomultiplier tube, and Clan-Thompson attachments. Flavin M ) , in 0.01 M sodium phosphate rates for decomposition of the free bases were slightly buffer at pH 7.5. was diluted with glycerol-water solutions greater than that of the Ncr-substituted analogues. to make 2 x 10- M flavin in 2 x 10- M buffer, with glyA previous study (Johnson and McCormick. 1973), cerol varying from 59-84 per cent by weight. The procedure indicating that the imidazole ring of the flavinyl histiand calculations were as described by MacKenzie er al. dines is partially protonated at pH 7. prompted the (1969). investigation of flavin-sensitized photooxidation of histidine at pH 8 and 2S°C, as shown in Fig. 1. As RESULTS AND DISCUSSION expected, the rates of oxidation of histidine by interArrobic photochemistry. The rates of flavin-sensit- molecularly added 10-w-carboxyalkylflavins (J2 and ized photooxidations of histidine and its derivatives 14) are only slightly greater at pH 8.0 than at pH 7.0. were determined by measuring the disappearance of The rates of histidine decomposition in the case of the the imidazole residue. This was done by diazotizing the flavinyl histidine amides (112 and 11,) are much slower histidine with Pauly's reagent and estimating the loss than the intermolecular cases with I,, I,, and 1,. This of color at 550 nm. The essence of this assay is that the curtailment in rate for the amide cases may, in part, rereagent must be much more sensitive to the histidine flect intramolecular, ground-state complex formation moiety than to the oxidation products. This assump- between flavin and histidine. Such complexing detion was justified by previous workers (Tomita et al., creases the fraction of flavin in excited forms capable 1969),who studied the photooxidations of N-benzoyl- of initiating the photooxidation of the histidine moiety. histidine and histidine as sensitized by rose bengal, A fluorescence and PMR study (Johnson and McCormethylene blue, and riboflavin. mick, 1973), in which the quenching of the 520-nm flaThe ability of several flavins to photosensitize inter- vin fluorescence emission and the chemical shifts of the molecularly the oxidation of histidine and its deriva- imidazole and isoalloxazine ring protons were studied tives at pH 7 and 25°C was measured. The rates of at various pH values and temperatures, definitely reactions for F M N and the 10-w-carboxyalkyltlavins showed some nonfluorescent, ground-state complex with histidine are similar, but F M N is 4 times more formation. Furthermore, this particular type of intereffective a sensitizer than FAD. This diminished pho- action is greater for the short-chain flavinyl histidine toreactivity of FAD can be attributed to intramolecu- (11,) than for the longer-chain one (IJs). Examination lar complex formation between the adenine and flavin of the photooxidation data in Fig. 1 further substanmoieties (Wu and McCormick, 1971; Weber, 1950; tiates the conclusions drawn from the fluoresccnce and McCormick et al., 1967). The flavin acid (I,) sensitizes PMR data, i.e. the rate of decomposition of the shortthe photooxidation of the histidine derivatives, i.e. chain amide (Ilz) is slower than the long-chain one N-acetyl-L-histidine and histidine methyl ester dihydro- (I15). Although not shown graphically, the rates of chloride, to a slightly greater degree than it does the photooxidation of amides 11, and 11, lie between free amino acid. After 3 h of illumination, 53 per cent those of 11, and 11,. The rates are in the following of the histidine remains, compared to only 44 per cent decreasing order: 115 > 11, z 11, > [Iz. of the N-acetyl derivative. This differs from results The extent of ground-state complex formation can obtained for the intermolecular FMN-sensitization of be semiquantitatively estimated using fluorescence tryptophan, tyrosine, and their N-acetyl derivatives polarization. The double-reciprocal plot of polariza(Wu and McCormick, 1971). In the latter cases, the tion of fluoresence of an intermediate-length flavinyl
Research Note
201
I
0
2
4
6
8
0
10
1 '7)
1
I I I 3 4 5 Hours of Illumination I
2
6
Figure 2. Double-reciprocal plot of polarization of fluorescence of flavinyl histidine (11,) and free flavin (I,) at pH 7.5 and 22°C in aqueous glycerol solutions of varying viscosity.
Figure 3. Anaerobic photolysis of histidine and derivatives at pH 8. Aqueous solutions contained 8 x 10- ' M flavinyl histidine (112,5) or flavin and histidine in 005 M sodium phosphate buffer (25°C).
histidine (11,) and flavin acid (I4) a t pH 7.5 in aqueous glycerol solutions of varying viscosity is shown in Fig. 2. From the ratio of the slopes (1-8), which is directly proportional to the ratio of mean lifetimes, and from the ratio of fluorescence of free flavin to flavin amide, which was determined as 2.8, it can be calculated (MacKenzie et al., 1969) that approximately 80 per cent of the quenching in 11, is due to ground-state complex formation. The simple demonstration of intramolecular complexing for the amide does not unequivocally show that such of the flavin moiety as becomes excited is unable to photooxidize the histidine. It may be that some of the excitation energy is dissipated by collisional interactions. There isalso the possibility that the imidazole and isoalloxazine rings are not as frequently mutually oriented in such a manner as to give proper oxidative interaction. This latter possibility seems unlikely, since the additional decomposition of histidine added to the amides is rather slight. These results tend to support the tenet that ground-state complexing impairs formation of the light-excited flavin, which may additionally be reverted to ground state by nonphotochemically productive collisions. Hence, decrease in the photooxidation of histidine within the amides is not mainly attributable to steric factors. The effect of intermolecularly added flavin on the photooxidation of histidine within flavinyl histidine was also investigated. The rates of decomposition of the mixtures of flavinyl amide and flavin (11, and 1,; 115 and I,) at pH 8 are much greater than for amide alone. For example. after 3 h of illumination, there is twice as much histidine remaining in the case of 11, as compared to the same amide to which equimolar 10-0cdrboxypentylflavin (I5) has been added. Further comparison shows that the rate of photooxidation is greater for the case of histidine and flavin (I,) than for flavinyl histidine (1I2,,) and flavin (12,5J. This suggests that the histidine within the amide is less accessible to added free Havin than that in a mixture of flavin plus histidine. The further importance of stereochemistry
can also be appreciated by the fact that the rate of decomposition of the larger and more bulky amide (115 15) is slower than the smaller one (11, I,). This agrees with similar measurements made on flavinyl tryptophans and tyrosines (Wu and McCormick, 1971). Previous work (Johnson and McCormick, 1973), in which the fluorescence-pH profiles of the flavinyl histidines were studied, showed that a t p H 4 and below there were very few isoalloxazine-imidazole interactions. This prompted the investigation of the rates of flavin-sensitized photooxidations of histidine at pH 4.0. The equimolar mixture of histidine and either FMN or 10-w-carboxybutylflavin (I4) shows a much slower degradation rate at pH 4 than at pH 7 or 8. This result agrees with a previous study (Taylor and Radda, 1971), which indicated very little photooxidation of histidine when the imidazole ring is protonated. The rates of photooxidation of flavinyl histidines (112.5) are lower at pH 4 than a t pH 8. The intermolecular addition of histidine or FMN to the amides has little effect on the rates. Thus, in the case of the flavinyl histidines, not only do the histidine and flavin moieties show very little association at p H 4 (Johnson and McCormick. 1973), but the ability of the flavin to sensitize the photooxidation of the protonated imidazole ring is also markedly decreased. The effect of temperature on photooxidation of the flavin amides follows an optimum. The increase in rate upon elevating the temperature above 25°C is probably due to greater dissociation of the ground-state complex, allowing the 450-nm light to create more triplet flavin. However, as the temperature increases, so do the molecular collisions, which causes quenching of the excited state. This probably explains why there is a decrease in rate when going from 35 to 50°C. Anaerobic photochemistry. The anaerobic oxidation of amino acids to keto acids and ammonia by lightexcited flavin provides a close analogy to amino acid oxidase systems. Byrom and Turnbull (1970) propose that triplet flavin abstracts hydrogens from the amino
+
+
208
A. P. BELLand D. B. MCCORMICK P. G. JOHNSON,
acid substrate, leading to production of dihydroflavin and concomitant loss of the yellow color. On admission of oxygen into the reduced solution, the oxidized flavin quantitatively reappears. The per cent loss of absorbance at 445 nm, when flavin solutions are irradiated anaerobically a t pH 8, is shown in Fig. 3. The rate of such color loss in the case of F M N plus histidine is much faster than for 10-w-carboxybutylflavin (I,) and histidine. In fact, after 6 h of illumination in the absence of oxygen, there is twice as much oxidized flavin in the I, case than in the F’MN one. However, if oxygen is then admitted to the systems, 92 per cent of the color returns to the histidine solution containing 14, while only 78 per cent of the color returns to that containing FMN. These data seem to indicate that a considerable amount of the F M N undergoes a photobleaching reaction. Anaerobic photobleaching includes a series of reactions in which both the excited singlet and triplet states of the flavin (Song and Metzler, 1967) intramolecularly react with its side chain to give various products, including lumiflavin and lumichrome. Thus, upon admission of air to such a system, only partial restoration of the absorbance (445 nm) is observed. Apparently, the greater part of photobleaching in the cuvette with FMN is due to the presence of the 2’-hydroxyl in the ribityl chain (Moore et al., 1963). Some photobleaching occurs in the I,-histidine system. It is known (Yang and McCormick, 1965) that the presence of a hydroxyl group on the 2’-position of the side chain is optimal, but not necessary, for intramolecular reaction with the isoalloxazine ring.
The anaerobic photolysis of flavinyl histidine amides (II2 and 115) produces less than 5 per cent loss in color, even after 6 h of irradiation at pH 8 and 25°C. This is to be expected, since the reactive amino groups necessary for ammonia production exist as amides in these particular compounds. Although not shown graphically, 112 and 11, retained greater than 90 per cent of their absorbance at 445 nrn after 24 h of constant illumination under anaerobic conditions (pH 8). Upon addition of oxygen, the absorbance returned to only 92 per cent of the original in both cases. As measured quantitatively using Pauly’s reagent, there was no apparent destruction of the imidazole moiety for either 112 or 115. This resistance to photobleaching, and the fact that the short-chain amide reacts slower than the long-chain one, seems again to suggest intrarnolecular, ground-state complex formation between the isoalloxazine and imidazole moieties. This interaction decreases formation of excited-state flavin necessary for initiation of photobleaching reactions. The effect of intermolecularly added L-histidine on the photoreduction and photobleaching of flavin in 112 and 115 is also depicted in Fig. 3. The rates are slightly higher than for the flavinyl amides alone. Admission of air to the IIs plus histidine system after 6 h of illumination returns the color to 99 per cent of its original value. These data seem to indicate that photoreduction, rather than photobleaching, is operating in these cases, and the lower rate is again due mostly to complexation of isoalloxazine and imidazole.
REFER EN CES
B y r o n P. and J. H. Turnbull (1968) Photochem. Photobiol. 8, 243254. Fischer, E. and L. H. Cone (1908) Ann. Chem. 363, 107-117. Fory, W., R. E. MacKenzie and D. B. McCormick (1968) J . Heterocy. Chem. 5,625-630. Frisell, W. R., C. W. Chung and C. G. MacKenzie (1959) J . Biol. Chem. 234, 1297-1302. Galston, A. W. (1950) Science 111,619-624. Johnson, P. G. and D. B. McCocmick (1973) Biochemistry 12,3359-3364. MacKenzie, R. E., W. Fory and D. B. McCormick (1969) Biochemistry 8, 1839-1844. McCormick, D. B. (1970) Experientia 26, 243-244. McCormick, D. B., J. F. Koster and C. Veeger (1967) Eur. J. Biochem. 2,387-391. Mohler, H., M. Briihmiiller and K. Decker (1972) Eur. J. Biochem. 29, 152-155. Moore, W. M., J. T. Spence, F. A. Raymond and S. D. Colson (1963) J . Am. Chem. SOC.85, 3367-3372. MWler, F., P. Hemmerich, A. Ehrenberg, G. Palmer and V. Massey (1970) Eur. J. Biochem. 14, 185-196. Patek, D. R. and W. R. Frisell (1972) Arch. Biochem. Biophys. 150,347-354. Penzer, G . R. (1970) Biochem. J. 116,733-743. Song, P A and D. E. Metzler (1967) Photochem. Photobiol. 6,691-709. Tabor, H. (1957) In Merhods in Enzymology(Edited by S. P. Colowick and N. 0. Kaplan), Vol. 3, pp. 63@-631. Academic Press, New York. Taylor, M.B. and G. K. Radda (1971) In Methods in Enzymology (Edited by D. B. McCormick and L. D. Wright), Vol. 18(B), p. 496. Academic Press, New York. Tomita, M., M. Irie and T. Ukita (1969) Biochemistry 8, 5149-5160. Tu,S.-C. and D. B. McCormick (1973) J . B i d . Chem. 248,633%6347. Walker, W. H. and T. P. Singer (1970) J. Biol. Chem. 245, 42244225. Weber, G. (1950) Biochem. J . 47, 114-122. Wu, F. Y.-H. and D. B. McCormick (1971) Biochim. Biophys. Acra 236,479-486. Yang, C. S.and D. B. McCormick (1965) J. Am. Chem. SOC.87,57635765.
