The porphyrin-sensitized photooxidation of horseradish apoperoxidase

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

172, 565-573

(19761

The Porphyrin-Sensitized Photooxidation Apoperoxidase’ YEOU-JAN Department

of Biology,

KANG2 University Received

JOHN

AND

of Utah, July

Salt

of Horseradish

D. SPIKES Lake

City,

Utah

Ml12

28, 1975

Horseradish apoperoxidase (apoHRP) was reconstituted with various porphyrin derivatives, e.g., ferric, cupric, manganese, and zinc protoporphyrin IX, metal-free protoporphyrin IX, hematoporphyrin IX and deuteroporphyrin IX. The visible absorption spectra of these porphyrin-apoHRP complexes were examined. The time required for maximum development of the new Soret peak after reconstitution was used to measure the rate of porphyrin-apoHRP reconstitution. All of the four metal-protoporphyrins reconstituted with apoHRP at the same rate as metal-free protoporphyrin IX, whereas, for the metalfree porphyrins, the rates of reconstitution were in the order of deuteroporphyrin IX > hematoporphyrin IX > protoporphyrin IX. The porphyrins on the reconstituted porphyrin-apoHRP complexes were used as localized photosensitizers for photodynamic studies. No amino acid residues were oxidized on illumination of the ferric, cupric and manganese protoporphyrin IX-apoHRP complexes due to the paramagnetic properties of these metal ions. With diamagnetic zinc ion, two histidine and one methionine residues were oxidized which was the same as in the protoporphyrin IX- and hematoporphyrin IX-apoHRP complexes. However, only one histidine was destroyed on illumination of the deuteroporphyrin IX-apoHRP complex. The results confirmed the resistance of horseradish peroxidase to photodynamic action and suggested the involvement of at least one histidine residue in the heme environment of horseradish peroxidase.

Most enzymes are inactivated on illumination in the presence of suitable photosensitizers; inactivation results from the sensitized photooxidation of the side chains of cysteine, histidine, methionine, tryptophan and tyrosine in the protein. This phenomenon is often termed photodynamic inactivation (1). The photooxidation of the protein can be limited to highly localized regions by using sensitizers covalently coupled or bound at specific sites on the molecule; under these conditions, only those susceptible residues located within a few angstrom units of the bound sensitizer are photooxidized (2-5). Residue orientation as well as distance may also be involved in this kind of selective photooxidation. This ’ Supported by the U. S. Energy Research and Development Administration under Contract No. E(ll-l)-875. 2 Present address: Marrs McLean Department of Biochemistry, Baylor College of Medicine, Texas Medical Center, Houston, Tex. 77025. Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

method has been used in a number of recent studies since it permits at least a limited probe of the three-dimensional structure of proteins in solution. Hemoproteins are of particular interest in studies of localized photooxidation since the heme porphyrin moiety, which is bound at a specific site on the molecule, represents a potential endogenous photosensitizer. Three strategies have been used to exploit this situation. The first involves using hemoproteins in which the heme is naturally in a low spin state; such porphyrins are efficient sensitizers. In contrast, hemes with iron in a high spin state (as is characteristic for most hemoproteins) are not sensitizers and in fact act as protective agents against photooxidation reactions. Jori et al. (2) showed that illumination of horse heart ferricytochrome c (in which the heme is naturally in a low spin state) at pH 8.2 led to the selective photooxidation of histidine-18 and methionine-80 565

566

KANG

AND

which are known to be ligands for the iron in this protein. With ferrocytochrome c, tryptophan-59 was photooxidized in addition suggesting that this side chain is closer to the iron atom in the ferro derivative (41. The second strategy involves converting a high spin ferroprotein to a low spin form prior to illumination. This approach has been used by Folin et al. (6) in studies on horse and sperm whale ferrimyoglobins which are insensitive to light. Ligation of the myoglobins with CN- gave low spin derivatives in which the histidine-93 residues were selectively photooxidized on illumination. Illumination of the low spin CO and 0, derivatives gave selective photooxidation of histidine-93 and histidine-64 in sperm whale myoglobin; these residues, as well as methionine-131 were photooxidized in horse myoglobin. The third strategy involves replacing the hemoprotein heme with photosensitizing porphyrins which bind at the same site on the apoprotein. Usually, the reconstituted porphyrin-apohemoproteins have the same conformation as native hemoproteins and exhibit similar physical properties (7); therefore, photooxidation of porphyrinapohemoproteins gives another reliable approach for probing the three-dimensional structures of hemoproteins in their native forms. This technique was used by Breslow et al. (8) who found that illumination of a 1:l complex of protoporphyrin IX with sperm whale apomyoglobin initially photooxidized histidine-93 which is known to be located very close to the heme iron in cyrstalline myoglobin. Human globin complexed with protoporphyrin IX lost most of the histidine and two methionine residues on illumination (9). Mauk and Girotti (10) recently reported that illumination of a 1: 1 protoporphyrin-apoHRp complex resulted in the destruction of one histidine residue. The present paper described the binding of a variety of metal-containing and metal-free porphyrins to apoHRP and the selective photooxidation of amino acid 3 Abbreviations used: HRP, horseradish peroxidase, isozyme c; apoHRP, horseradish apoperoxidase; CM-cellulose, carboxymethylcellulose; DEAE-cellulose, diethylaminoethyl cellulose.

SPIKES

residues resulting from the illumination certain of these complexes. MATERIALS

AND

of

METHODS

Horseradish peroxidase (HRP) (EC 1.11.1.7) (Sigma Chemical Co., Type II, Lot ZOC-2820, R.Z. 1.37) was purified by chromatography on CM and DEAE-cellulose columns essentially according to the method of Shannon et al. (11). The fraction corresponding to isoperoxidase c was rechromatographed on CM-cellulose until the R.Z. value was greater than 3.0. After dialysis, this material was passed through a DEAE-cellulose column; the collected enzyme with an R.Z. value of 3.2 was dialyzed, lyophilized and stored at 0°C. The concentrations of HRP solutions were measured by their absorbance at 403 nm and by using a millimolar extinction coefficient of 95 rnM-‘cm-’ (12). Horseradish apoHRP was prepared by the removal of heme from HRP with cold acidbutanone by using Teale’s (13) method as moditied by Yonetani (14). The purified apoHRP was exhaustively dialyzed, lyophilized, and stored at 0°C. The concentrations of apoHRP solutions were measured by their absorbance at 277 nm and by using a millimolar extinction coefficient of 13 rnM-‘cm-’ (15). All spectral measurements were made in a Cary Model 14M spectrophotometer. Protoporphyrin IX (Calbiochem Co., A grade, Lot 001447) was used without further purification. Hematoporphyrin IX was a product of Fluka AG (Switzerland), and deuteroporphyrin IX was a gift from Dr. Bruce F. Burnham, Department of Chemistry and Biochemistry, Utah State University, Logan, Utah.’ Ferric protoporphyrin IX (hemin) was purchased from Calbiochem Co. (Lot 900765) and used without further purification. Cupric, manganese and zinc protoporphyrin IX were prepared by the reaction of metal-free protoporphyrin IX in glacial acetic acid with the acetate of the metal at 80°C as described by Fischer and Piitzer (161. Porphyrin-apoHRP complexes for photodynamic studies were prepared by mixing equal volumes of porphyrin solution and apoHRP solution of the same molarity and allowing the mixture to stand in the dark for at least 60 min at 25°C. The time course of complex formation was determined by recording the spectra of the mixtures at selected intervals. The porphyrin solutions were prepared by dissolving the compounds in the minimal volume of 0.1 M NaOH and diluting to the desired concentration with 0.1 M sodium phosphate buffer, pH 6.0. In some experiments, the mixtures were rapidly passed through a Sephadex G-25 column to remove uncomplexed porphyrin. Reaction mixtures for photodynamic treatment 4 Present address: Porphyrin 31, Logan, Utah 84321.

Products,

P. 0. BOX

PHOTOOXIDATION were prepared by mixing 1.0 ml of a solution of the complex (2 mg/ml) with 1.0 ml of 0.5 M sodium phosphate buffer, pH 8.0. The mixtures were placed in lo-dram plastic vials and illuminated with shaking in air in a 25 ? 1°C water bath with 150-W Sylvania reflector-flood lamps at a distance of 12 cm. Samples were removed at intervals for amino acid analysis by the method of Moore and Stein (17) by use of a Beckman Spinco Model 120C amino acid analyzer. Acid hydrolysis of enzyme samples was carried out under nitrogen in 6 N constant-boiling HCl in glass hydrolysis tubes for 22.h at 110°C. Methionine was measured following hydrolysis of samples for 22 h at 110°C under nitrogen with 4.5 N NaOH in plast,ic tubes. Tryptophan residues were determined after alkaline hydrolysis of samples in the presence of starch, as described by Hugli and Moore (18). RESULTS

Formation and Spectral Properties phyrin-apoHRP Complexes

of Por-

The purpose of these experiments was to examine the combining of several selected porphyrins with apoHRP in order to estnblish conditions for the preparation of porphyrin-apoHRP complexes to be used in photodynamic studies. Figure 1 shows the spectrophotometric titration of apoHRP

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PEROXIDASE

with hemin. ApoHRP preparation in this study was able to bind hemin in a 1:l molar ratio. Equal volumes of equimolar porphyrin solution and apoHRP were mixed as described above and allowed to stand in the dark at 25°C. At different time intervals, the spectra of the mixtures were recorded. A typical set of time-course absorption changes for hemin-apoHRP complex (=HRP) formation is shown in Fig. 2. As may be seen, hemin (dotted line in Figs. 2 and 3A) has a somewhat broad absorption peak at 375 nm (the Soret band); on mixing with apoHRP, the absorption peak progressively shifts to 403 nm, representing the formation of the heminapoenzyme complex (solid line in Figs. 2 and 3A). The absolute peak extinction coefficient of the complex is greater than that of hemin alone. Also, as may be seen, the complex has two additional absorption peaks at 498 and 640 nm. Complex formation is very fast during the first 2 min after mixing and is more than 95% complete 30 min after mixing. Complexes are also produced between other metalloporphyrins

I01 HemIn + ApoHRP

08

E 04

Hemln

0 0

2

Micramoles

+ ApoHRP

,

,

,

4

6

8

,

-1

10 12 h%mln added / 5 mlcromoles ApoHRP

FIG. 1. Spectrophotometric titration of horseradish apoperoxidase (apoHRP) with hemin. The reaction mixture consisted of 3.0 ml of apoHRP (0.8 mg/ml = 20 @mol/ml) and 3.0 ml of 0.5 M sodium phosphate buffer, pH 7.3. Aliquots, 0.5 ml, of the reaction mixture were mixed with 0.5 ml of different concentrations of hemin containing the amounts shown and incubated in the dark at 25°C for 30 min to permit reconstitution to occur, and the absorbance was measured at 403 nm.

02

0

320

400

500 Wavelength

600

c

inmi

FIG. 2. Time course of the spectral changes during the reconstitution of hemin (ferric protoporphyrin IX) with horseradish apoperoxidase (apoHRP) at pH 6 and 25°C. The reaction mixture contained 0.5 ml of 1 x lo-” M apoHRP in 0.1 M sodium phosphate buffer, pH 6.0.

568

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AND

and apoHRP as shown in Fig. 3. Manganese protoporphyrin IX (Fig. 3B), with a Soret peak at 420 nm, forms a complex with apoHRP with a peak at 425 nm; small peaks also appear at 522 and 624 nm. Similarly, zinc protoporphyrin IX (peak at 435 nm) gives a complex with the major peak at 456 nm and minor peaks at 526 and 580 nm (Fig. 3C), while cupric protoporphyrin IX (peak at 417 nm) gives a complex with peaks at 440, 536 and 610 nm (Fig. 3D). The time course for the formation of the porphyrin-apoHRP complex was essentially the same with all four metalloporphyrins. Similar studies were carried out with metal-free porphyrins. Figure 4A shows the results with protoporphyrin IX; as may be seen, the Soret peak shifts from

SPIKES IA I 0 t

0 8 0 6 0 4 0 2 0 I 0 0 8

I 2

Cc

300 Q- 6

hln-Protoporphyrln IXApoperoxidase

M;-Protoporphyrm

IX

Zn-Protoporphynn Apopemridase

300

400

IX-

Zn-Protoparphyrin

IX

Cu-Protoporph,wn Apoperox~dase

IX-

Cu,-Protoporphyrl”

IX

SO0 WAVELENGTH

600

400

500 WAVELENGTH

600

J 100

mm)

FIG. 4. Absorption spectra of metal-free porphyrins and of metal-free porphyrin-apoperoxidase complexes. Reaction conditions were the same as described for Fig. 3. Free porphyrins used were protoporphyrin IX (A), hematoporphyrin IX (B) and deuteroporphyrin IX (Cl.

I

t

n

I

(ram)

FIG. 3. Absorption spectra of metalloporphyrins and of metalloporphyrin-apoHRP complexes. The reaction mixtures contained 0.5 ml of 1 X 10-j M horseradish apoperoxidase and 0.5 ml of 1 x lo-” M ferric protoporphyrin IX [A), manganese protoporphyrin IX (Bl, zinc protoporphyrin IX (Cl, or cupric protoporphyrin IX CD). All solutions were 0.1 M sodium phosphate buffer, pH 6.

368 nm for the porphyrin to 398 nm for the protoporphyrin IX-apoHRP complex. Four minor peaks occur in the visible rehematoporphyrin IX gion. Similarly, shows a shift from 390 to 404 nm in the formation of the complex (Fig. 4B). Deuteroporphyrin IX represents a slightly more complicated case since even in very low concentrations (25 PM at pH 6.0) it appears to be present largely in the form of dimers or polymers (B. F. Burnham, personal communication) with the Soret peak at approximately 360 nm; at lower concentrations, in the monomer form, the absorption peak is at 390 nm (this appears as a shoulder in Fig. 40. In the present experiment, then, as may be seen in Fig. 4C, there is a shift in the position of the main peak from approximately 363 to 398 nm for the complex. There is also a twofold increase in the absorbance of the Soret peak

PHOTOOXIDATION

OF

and significant increases in the four minor bands at approximately 496, 533, 551, and 603 nm. The formation of the deuteroporphyrin IX-apoenzyme complex was quite fast; maximum development of the new absorption peak occurred in about 10 s. The times required for 95% formation of the complex with hematoporphyrin IX and with protoporphyrin IX were approximately 20 and 30 min, respectively. Photooxidation Complexes

of

Porphyrin-apoHRP

Solutions of the complexes were illuminated and then hydrolyzed and subjected to amino acid analysis to determine which, if any, of the amino acid residues were photooxidized with the complexed porphyrin serving as the photosensitizer. The HRP molecule contains a number of photooxidizable amino acid residues (three histidine, three methionine, one tryptophan five tyrosine). Amino acid analyses of ferric, cupric, manganese and zinc protoporphyrin IXapoHRP complexes after being exposed to visible light for 30 min at pH 8.0 are shown in Table I. As may be seen, there is no destruction of amino acid residues in the ferric protoporphyrin IX complex. This conP 1. x . . ,I , .I. nrms earlier work snowing tnat tnis complex is completely insensitive to visible light even if the iron is in a low spin state (19). Complexes involving the other two paramagnetic metals, copper and manganese, were also completely insensitive to visible light In contrast, illumination of the zinc protoporphyrin IX-apoHRP complex (zinc is diamagnetic) resulted in the rapid destruction of one methionine and TABLE AMINO

ACID

ANALYSES

-

Photosensitive acid Histidine Methionine Tryptophan Tyrosine

amino

two histidine residues; no tryptophan or tyrosine residues were destroyed (see last column of Table I). The zinc derivative thus behaves much like metal-free protoporphyrin IX in that, after 30 min of illumination, it gives a similar pattern of amino acid destruction on apoHRP as described below. Similar experiments were also carried out with complexes of the metal-free porphyrins except that samples were taken at intervals during illumination in order to examine the time course of amino acid residue destruction. Figure 5 (closed symbols) shows the time course for the photooxidation of amino acid residues in the protoporphyrin IX-apoHRP complex on illumi-

f

t! i z g 4 5

40

20

. . . . Protowphyrm apoperoxldose

IX-horseradish

o Ao ~ Hematoporphyrln apoperoxldase 0

IX-horseradish

20

10 Time

of illumination

30

40

(mln)

FIG. 5. The time course of amino acid destruction in the protoporphyrin IX-apoperoxidase (apoHRP1 complex (closed symbols) and the hematoporphyrin IX-apoHRP complex (open symbols) on illumination with visible light in air at 25°C. The reaction mixtures contained 1.0 ml of porphyrin-apoHRP complex solution (2 mglml) and 1.0 ml of 0.5 M sodium phosphate buffer, pH 8.

I

OF METALLOPROTOPORPHYRIN-HORSERADISH MIN OF ILLUMINATION” -

Dark

569

PEROXIDASE

control

AP~PEROXIDASE

Protoporphyrin

COMPLEXES

AFTER

30

IX

-

3.1 3.0 1.2 5.2

Ferric

Cupric

3.0 3.1 1.2 5.3

3.2 2.9 1.3 4.8

D The reaction systems contained 1.0 ml of metalloprotoporphyrin and 1.0 ml of 0.5 M sodium phosphate buffer, pH 8. Illumination

IX-apoHRP was carried

Manganese 3.0 3.1 0.9 4.9 complex solution out in air at 25°C.

Zinc 1.1 2.0 1.1 5.0 (2 mg/ml)

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KANG

AND

nation. As may be seen, 65% of the histidine and 33% of the methionine residues are destroyed after only 5 min of illumination, representing the destruction of one methionine and two histidine residues per molecule. Amino acid destruction during this time is first order in amino acid concentration as is usually found in photodynamic systems (1). Essentially no further destruction of histidine and methionine residues occurred during the next 35 min of illumination. Tyrosine and tryptophan residues are not affected during the period of illumination used. Illumination of the hematoporphyrin IX-apoHRP complex gave essentially identical results (Fig. 5, open symbols). In contrast, illumination of the deuteroporphyrin IX-apoHRP complex caused the rapid destruction of only a single histidine residue; none of the other photosensitive amino acid residues was affected (Fig. 6). Prolonged illumination of all three of the metal-free porphyrin-apoHRP complexes destroyed additional amino acid residues. As shown in Table II, 3 h of illumination gave a further destruction of histidine and methionine residues in all three complexes; there was also a destruction of tryptophan and tyrosine residues. DISCUSSION

The reconstitution of apoperoxidase with its prosthetic group, heme, to restore the original enzymatic activity has been studied extensively with both HRP (20-22) and cytochrome c peroxidase (14). ‘However, relatively few studies have been made of the reconstitution of apoperoxi-

FIG. 6. The time course of amino acid destruction in the deuteroporphyrin IX-apoperoxidase complex on illumination with visible light. The reaction conditions were the same as for Fig. 5.

SPIKES TABLE

II

AMINO ACID ANALYSES PORPHYRIN-HORSERADISH COMPLEXES -

Photosensitive amino acid

3 H OF ILLUMINATION”

AFTER --

Dark control

OF METAL-FREE APOPEROXIDASE

-

Complexes of horseradish apoperoxidase with Protoporphyrin

Histidine Methionine Tryptophan Tyrosine (I Reaction Table I.

3.0 3.2 1.1 5.1 __conditions

Hematoporphyrin

0.3 0.9 0.4 2.3 were

0.2 0.5 0.3 3.3 the

Deuteroporphyrin 0.5 1.2 0.6 2.9

same

as

for

dase with other porphyrin derivatives. Early studies by Gjessing and Sumner (23) only showed that the derivatives prepared by reconstituting apoHRP with cobalt, manganese,” nickel and copper protoporphyrin had little enzymatic activity; the spectral and other physical properties of the reconstituted complexes were not studied. Recently, Yonetani and Asakura (24) reconstituted manganese protoporphyrin IX with a variety of apohemoproteins and were also able to crystallize manganese protoporphyrin IX-cytochrome c apoperoxidase. The reconstitution of protoporphyrin IX with apoHRP has recently been reported (10); however, no studies have been reported on the reconstitution of apoHRP with other metal-free porphyrin derivatives until the present study. The absorption spectra of protoporphyrin IXapoHRP complex and manganese protoporphyrin IX-apoHRP measured in this study were found consistent with previously reported results (10, 24). Metal-free protoporphyrin IX and ferric, manganese, zinc and cupric protoporphyrins were found to combine with apoHRP at about the same rate in 0.1 M sodium 5 Gjessing and Sumner (23) showed that reconstituted manganese protoporphyrin IX-apoHRP exhibited 28% of peroxidase activity as compared to native HRP in one out of five experiments. However, this could not be repeated by Theorell (21). Recently, Yonetani and Asakura (24) crystallized this complex and claimed it was enzymatically active. In the present study, the enzymatic activities of these complexes were not examined.

PHOTOOXIDATION

phosphate buffer at pH 6.0. This suggests that the metal atom has little to do with the interaction of a porphyrin with apoHRP to give the complex. However, further experimental evidence is necessary to afirm it. The different metal ions in the protoporphyrin IX had little, if any, effect on the rate of reconstitution with apoHRP. The stoichiometric kinetics of all these reconstitutions, therefore, probably occur in the same kind of two-stage scheme as suggested for heme and apoperoxidase (22, 25). Similar results have also been observed with other hemoproteins (8, 9, 26-28). Differences in the rates of the reconstitution between heme protoporphyrin IX with apoperoxidase have been observed (10, 29); however, the rate of the complex formation is known to be quite pH dependent. The reaction rate of metal-free porphyrin-apohemoprotein complex formation is accelerated at lower pH whereas the reaction rate between the apoenzyme with ferric porphyrin was accelerated by higher pH (30). In addition, the dissociation of porphyrin aggregates into monomers at pH 6 would also affect the rate of porphyrin-apoenzyme reconstitution. Therefore these may account for the difference in the rates of reconstitution observed in the present work, carried out at pH 6.0, as compared with earlier work, carried out at pH 9 (10, 29). The present work shows that the rate of reconstitution depends strongly on the porphyrin side chains with deuteroporphyrin IX complexing with apoHRP very rapidly, hematoporphyrin IX much more slowly, and protoporphyrin IX even more slowly. All three of these porphyrins have propionic acid groups at the 6- and ‘7-positions, a primary structural requirement in order for a porphyrin to bind to apoHRP (31). However, the side chains at the 2- and 4-positions differ and may play an important role in the orientation of porphyrins into the heme “pocket” on the protei.n. The rather bulky groups in the 2- and 4-positions of protoporphyrin IX might sterically hinder the fitting of this porphyrin into the heme pocket while the much smaller hydrogen atoms in these positions on deuteroporphyrin IX may exert little steric hinderance. Differences in the

OF

PEROXIDASE

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side chains on the 2- and 4-positions of ferric protoporphyrin IX were also found to alter peroxidase activity in the reconstituted complexes (23, 31). Therefore, the 2-, 4-side chains on the porphyrin ring were suggested to be essential in the reconstitution of porphyrin and apoHRP. The insensitivity of the ferric protoporphyrin IX-apoHRP complex (=HRP) to visible light further supports previous findings that peroxidase is resistant to photodynamic inactivation (32, 33) due to the presence of the paramagnetic ferric ion. It has been known (34) that ferric and cupric hematoporphyrins do not act as photodynamic sensitizers in solution; the paramagnetic metal in these porphyrins markedly increases the efficiency of intersystem crossing from the singlet to the triplet states but also causes an almost lOOO-fold increase in the rate of the internal deactivation of the triplet states. Since photooxidations sensitized by porphyrins are mediated by the triplet state (34-361, the incorporation of a paramagnetic metal into the porphyrin would thus give a very ineffcient sensitizer. In fact, flash photolysis measurements on porphyrins containing paramagnetic metals show no detectable production of triplet states (37; unpublished results, this laboratory). Therefore, upon the illumination with visible light, none of the photosensitive amino acid residues in ferric, cupric, or manganese protoporphyrin-apoHRP complexes was destroyed. The diamagnetic metal porphyrins usually show good concentrations of triplet states in flash photolysis studies (38, 39; unpublished results, this laboratory). Thus, as would be expected, illumination of the zinc protoporphyrin IXapoHRP complex resulted in the photodynamic destruction of amino acid residues (two histidine, one methionine). Gennari et al. (34) found that both zinc and magnesium hematoporphyrins were good photodynamic sensitizers in solution. With all three metal-free porphyrinapoHRP complexes, at least one histidine residue is rapidly destroyed on illumination. This suggests strongly that at least one histidine residue must be located very close to the porphyrin-binding site on the

572

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protein. Only one histidine residue is photooxidized rapidly in the deuteroporphyrin IX (H atoms in the 2- and 4-positions) complex, while one methionine residue and two histidine residues are rapidly destroyed in the hematoporphyrin IX (hydroxyethyl groups in the 2- and 4-positions) and protoporphyrin IX (vinyl groups in the 2- and 4-positions) complexes. This suggests that deuteroporphyrin binds in such a way that only one histidine residue is within its sensitizing range. The other two porphyrins, with their more bulky substituents, may bind at a slightly different location such that an additional histidine residue and a methionine residue are close enough to be photooxidized. Although Mauk and Girotti (10) found that only one histidine residue was destroyed on the illumination of protoporphyrin-apoHRP complex, the reason for this difference is not known; it could be due to the difference in the reaction conditions used, such as the different pH, buffer systems or light intensity. The less specific pattern of amino acid residue destruction (Table II) on prolonged illumination may result from a release of the porphyrin as a consequence of the photodynamic destruction of amino acid residues essential to the heme-binding site; the released sensitizer could then diffuse into other regions of the molecule and sensitize the photooxidation of additional amino acid residues (8). Although all three of these metal-free porphyrins are efficient photodynamic sensitizers in solution (401, the destruction of different amino acid residues in the photooxidaton (Table II; Figs. 5 and 6) could also be attributed to different photosensitizing activities of these porphyrins since different side chains can apparently change the photosensitizing properties of the porphyrins (34). Although the involvement of singlet oxygen in the protoporphyrin IX-sensitized photooxidation has been suggested (9, 411, the detailed reaction mechanisms with respect to each individual porphyrin require further studies. REFERENCES 1. SPIKES, J. D., AND LIVINGSTON, vances in Radiation Biology

R. (1969) in Ad(Augenstein, L.,

SPIKES

2. 3.

4.

5 6

Mason, R., and Zelle, M., eds.), Vol. 3 pp. 29113, Academic Press, New York. JORI, G., GENNARI, G., GALIAZZO, G., AND SCOF FONE, E. (1970) FEBS L&t. 6, 267-269. JORI, G., GALIAZZO, G., MARCHIORI, F., AND SCOFFONE, E. (1970) Znt. J. Protein Res. 2, 247-256. JORI, G., GENNARI, G., FOLIN, M., AND GAG IAZZO, G. (1971) Biochim. Biophys. Acta 229, 525-528. SPIKES, J. D., AND MACKNIGHT, M. L. (1970) Ann. N. Y. Acad. Sci. 171, 149-162. FOLIN, M., GENNARI, G., AND JORI, G. (1974) Photochem. Photobiol. 20, 357-370.

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