Voh 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ] 209-I 2"15
March 31, 1992
RECONSTITUTION OF APOMYOGLOBIN WITH EXTENDED BILIVERDINS
Marcelo Femfmdez, Rosalia B. Frydman, Sara Bad and Benjamin Frydman Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, Buenos Aires, Argentina
Received February 3, 1992
SUMMARY: An analysis of the reconstitution of biliverdins with extended conformations and horse heart apomyoglobin was carded out. Biliverdins with the 5Z-syn, lOZ-syn, 15Z-anti and 5Z-anti, lOZ-syn, 15Z-anti conformations, as well as biliverdins with the Z,Z,Z, all-syn conformation recombined with apomyoglobin. In every case the P enantiomers were bound in excess to the M enantiomers, with exception of the 5-syn, l O-syn,15-anti biliverdin where the M enantiomer bound preferentially to the protein. Biliverdins with an anti conformation at the C-10 meso bridge did not recombine with the protein. It was concluded that the presence of a syn conformation at the C-10 methine conferred to the biliverdin the necessary helicity to fit into the apomyoglobin heme pocket. This regioselectivity is of importance in view of the well known analogy between the ligand domains of myoglobin and the C-phycocyanins. ®1992 Academic Press,
Inc.
Bile pigments interact with proteins in two different ways. In the phycobiliproteins as well as in phytochrome they are covalently bound to the proteins by one or two thioether linkages and the protein enforces extended (or stretched) conformations on the bilitrienes as a result of electrostatic interactions between the propionate groups of the ligand and the basic residues of the proteins (1, 2). In the insect biliproteins known as insecticyanin (3) and biliverdin binding protein (BBP)(4), the biliverdin prosthetic groups are not covalently bound to the protein but interact with the latter through electrostatic and hydrophobic forces. In these biliproteins the biliverdins retain their energetically favored helical conformations. In the case of the phycobiliproteins, the biliverdins are derived from biliverdin IX t~ while in the insect biliproteins they are of the IX y type. It has been shown that apomyoglobin and the o~ and [3 subunits of C-phycocyanin share a similar "globin fold"; although their primary and secondary structures are quite different (5). This remarkable similarity led Falk and coworkers to analyze the recombination of sperm whale apomyoglobin with a number of helical biliverdins (6, 7). They found that apomyoglobin recombined with the biliverdins as long as the latter carded one or more propionate residues which allow them to anchor on the basic residues (His 97 and Arg 45) located at the entrance of the heme pocket. Their results led them to conclude that the apomyoglobin pocket is indifferent with respect to the steric and functional patterns present in the biliverdins (7). Since in the phycobiliproteins the biliverdins have extended conformations, recombination studies of apomyoglobin with extended
1209
0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
biliverdins are relevant to clarify if indeed steric factors are important for the interaction between the heme pocket of apomyoglobin and the biliverdins. An analysis of the binding of biliverdins with extended conformations to apomyoglobin should reveal to which extent the heme-binding domain of the latter and those present in the phycobiliproteins are analogous. We therefore explored the recombination of apomyoglobin and synthetic extended biliverdins with different degrees of stretching and with different configurations at the meso-bridges. The results showed that steric factors are crucial for the recombination of the biliverdins with the apomyoglobin and that the heme pocket in the latter is highly regioselective toward the conformation of the ligand.
MATERIALS AND METHODS Materials. Biliverdins IX ct l a , IX [3 l b , IX 7 lc and IX 8 l d (Fig.l) were obtained by the coupled oxidation of heme IX and were separated as described elsewhere (8). Biliverdins 2-6 (Fig. 1) were prepared by synthesis (9). Apomyoglobin was prepared by the acid-butanone method (10) from horse heart myoglobin (Sigma). All the reagents and solvents were of the highest analytical grade. PD-10 (Sephadex G-25) columns were from Pharmacia. Methods. In a typical preparation of an apomyoglobin-biliverdin complex, 15 mg of horse heart myoglobin were dissolved in 2 ml of 20 mM phosphate buffer, pH 6.0; the solution was adjusted to pH 2.8 with HC1, and the acid solution was extracted thrice with an equal volume of butanone. The berne-less apomyoglobin was dialyzed overnight against 20 mM phosphate buffer, pH 6.0. The apomyoglobin was then recombined with the biliverdins dissolved in a minimum volume of 0.1 N NaOH. The solution was adjusted to pH 6.0 with phosphate buffer. A ratio of 1:1.5 of protein to the chromophore was used. The mixture was allowed to stand for 2 h at 4 -0C and the
H
N--
R~
NH
N
,)7-NH
R4
N~-------~ 3_
Z
!
p
P~/N~ H
O.-
. ' ~ N ~ j ~ ,
p
£
5--~-X
5 P
l~r 0
_
6
~
_
N"
-0
P= CH2CH2CO2H
Figure 1. Structures of the biliverdins used in this study.la: biliverdin IX ¢t: Rl=R3=R6=R7=methyl; R4=R5=propionate; R2=R8=vinyl. l h : biliverdin IX ~: Rl=R4=R5=R7=methyl; R2=R3=propionate; R6=R8=vinyl. l c : biliverdin IX 3t: R2=R3=R5=R7=methyl; Rl=R8=propionate; R4=R6=vinyl. I t : biliverdin IX 8: Rl=R4=R6=R8=rnethyl; R2=R3=propionate; R5=R7=vinyl. 1210
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
complex was then isolated by filtration through a PD-10 column. The excess of biliverdin binds to the gel matrix and can be eluted with 0,1% Triton in the same buffer. When the dimethyl ester of biliverdin 2 was used for reconstitution, it was dissolved in the minimum volume of acetone so that upon mixing with apomyoglobin the final concentration of the acetone was less than 20%. After dialysis any excess of biliverdin ester was centrifuged off and the complex was filtered through the PD-10 column. Formation constants were calculated by quantitative interpretation of the visible difference spectra. In the case of biliverdin 2 we considered that e600 = e667 = 71.8 mM -1 cm -1. Protein concentrations were estimated by the method of Bradford (11) using bovine serum albumin as a standard. Optical spectra were measured with a double beam Hitachi U-2000 spectrophotometer. CD spectra were recorded with a JASCO J-20 Spectropolafimeter.
RESULTS Biliverdin IX o~ l a was recombined with horse heart apomyoglobin as described by Marko et al (6) for sperm whale apomyoglobin and essentially the same results were obtained. Horse heart and sperm whale myoglobin have identical heme cavities as revealed from their X-ray structures (11, 12) and from functional studies (13). The minor differences in the CD3 salt bridges of the apomyoglobin (Arg in sperm whale and Lys in horse heart myoglobin) should not affect their recombination properties with the biliverdins. The Cotton effect of the CD spectrum indicated that of the two helical enantiomers (P and M) of biliverdin IX c~ la, apomyoglobin binds preferentially to the P enantiomer, as reported by Marko et al. (6). Apomyoglobin was then recombined with biliverdins IX 13 lb, IX 7 lc and IX 8 ld. The UV/vis spectra of the complexes (Figs. 2A, 2C, and 2E) showed batochromic shifts in the short and long wavelength absorption bands. In the latter
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1-~75
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3.0 _
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0
400
~-~
"-/ ~
s S°---
400
.
t 368
•
600
800
4(30
Sl~)
~
-IO.O
700
Wavelength, nm
Figure 3. (A): electronic absortion spectra of biliverdin 2 ( ) and biliverdin 2-apomyoglobin complex (----). (B): CD spectrum of the biliverdin 2- apomyoglobin complex. All the spectra were recorded in phosphate buffer, pH 6.0. 1212
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.J%
0.6
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A
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-IO.O 200
400
600
800
400
500
660
[~ K)
Wavelength, nm
Figure 4. Electronic absortion spectra of biliverdin 3 (A), its dirnethyl ester (C) ( ), and of their complexes with apomyoglobin (----). The spectrum of biliverdin 3 dimethyl ester was recorded in acetone, while that of its complex with apomyoglobin was recorded in aqueous phosphate buffer, pH 6.0. CD spectra of biliverdin 3 (B) and biliverdin 3 dimethyl ester (D) apomyoglobin complexes.
the chromophores of the phycobiliproteins (1). Its visible absorption maxima suggests the presence of more than one conformational form, a fact that could result from different dihedral angle torsions at the C4-C5 and C9-C10 bonds. It recombines with apomyoglobin and the spectrum of the complex shows the usual batochromic shifts (Fig. 4 A, dashed line). The Cotton effect of its CD spectrum indicated a preferential binding of the M enantiomer (Fig. 4 B). It is the only biliverdin which has been recombined with apomyoglobin where a preferential binding of this enantiomer has been found. It stands in contrast with biliverdin IX 13where the P enantiomer binds preferentially (Fig. 2 B). Hence, the preference of the apomyoglobin pocket for the M enantiomer in 3 has to be attributed to the rigid anti configuration at C15. In the case of the dimethyl ester of 3 which also recombined with apomyoglobin (Fig. 4C) but only to ca. 15% of that of the free acid 3, the P enantiomer was the preferred one (Fig. 4 D). This fact underlines the great importance of the free propionate residues in the selective anchoring of one of the helical enantiomers in the myoglobin heme pocket. Biliverdins 4-6 (Fig. 1) did not recombine with apomyoglobin. This lack of recombination is noteworthy in biliverdin 4 where only the C10 double bond of the chromophore is part of an extended structure while the configurations at C5 and C15 keep the Z-syn chirality. The formation constants of the complexes of biliverdins IX (~ la, IX 13l b , IX y l e and IX 8 l d with apomyoglobin were found to be of the same order of magnitude (Table I). Among them the T isomer is the more readily complexed. In the case of biliverdin 2 (which is present in two conformations), two formation constants were calculated, one for each conformational form. The form absorbing at the longest wavelength had a formation constant thrice that of the other conformer (Table I). 1213
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Formation constants of the apomyoglobin-biliverdincomplexes Biliverdin
K ( M-1)
IX ~x la
3.4 x 105
IX [3 lb
1.6 x 105
IX y lc
8.0 x 105
IX8 ld
1.4x105
2 (617 nm)
4.0 x 105
2 (680 nm)
1.2 x 106
3
3.5 x 105
The constants were calculated as described in Materials and Methods.
DISCUSSION The obtained results confirm earlier data that the myoglobin heme-pocket is indifferent to the substitution type and pattern of the biliverdins (7). Changes in the former affect very little the complexation of the ligands with the protein. The results show, however, the critical importance of the steric factors for the complexation of the protein with the biliverdins. The lack of complexation of biliverdins 4-6 with apomyoglobin indicates the crucial importance of the configuration at the C10 bridge. When rings B and C are held in an extended conformation, no recombination with apomyoglobin takes place. Given the similarity of the "globin fold" of apomyoglobin and the phycocyanins, our results would explain why in the natural biliproteins of all types, the C10 bridge is always in the helically shaped
Z-syn configuration. Even the photodynamic equilibrium which
takes place in phytochrome affects only the isomerism at the C5 and C15 bridges (2). In the complexation of apomyoglobin with the four biliverdin IX isomers no major changes among them were found, either in the complexation constants or in the enantiomer discrimination. This suggests that the presence of biliverdin IX ot l a in the phycobiliproteins and of biliverdin IX y le in the insect biliproteins has a metabolic rather than a structural origin. A second factor which seems to be of relevance for the recombination of the biliverdins with apomyoglobin is the ability of the latter to preferentially bind one helical conformer. In every case when a biliverdin is bound to the apoprotein, a Cotton effect was detected in the CD spectra. In the more extended biliverdins (such as 4-6) no P/M isomerism is possible and this probably detracts from the flexibility needed by the ligand to fit into the protein pocket. The results reported above show how different the biliverdin binding domains of apomyoglobin (and therefore of phycocyanins) are from that of mammalian biliverdin reductase. The latter interacts with extended biliverdins to a much greater extent than with the helical ones (15). 1214
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS. This work was made possible by Grant GM-11973 from the National Institutes of Health (PHS). Support from CONICET (Argentina) is also acknowledged. We are indebted to Drs. H. Fern~indezand J.M. Delfino for recording the CD spectra. REFERENCES 1. Scheer, H. (1981) Angew. Chem. Int. Ed. Engl., 20, 241-261. 2. Riidiger, W. and Thiimmler, F. (1991) Angew. Chem. Int. Ed. Engl., 30, 1216-1228. 3. Riley, C. Y., Barbeau, B. K., Keim, P. S., Kezdy, F. J., Heinrickson, R. L. and Law, J. H. (1984) J. Biol. Chem., 259, 13159-13165. 4. Huber, R., Schneider, M., Mayr, I., MUller, R., Deutzmann, R.,Suter,F., Zuber H.,Falk, H. and Kayser, H. (1987), J. Mol. Biol., 198,499-513. 5. Schirmer, T., Bode, W., Huber, R., Sidler, W., and Zuber, H. (!985), J. Mol. Bio1.,184, 257-277. 6. Marko, H., MUller, N. and Falk, H. (1985), Monatsh. Chem., 120, 591-595. 7. Falk, H., Marko, H., MUller, N., Schmitzberger, W., and Stumpe, H. (1990), Monatsh. Chem., 121,893-901. 8. Frydman, R. B., Awruch, J. Tomaro, M. L., and Frydman, B. (1979),Biochem Biophys Res. Commun., 87, 928-935. 9. Iturraspe, J.B., Bari, S. and Frydman, B. (1989), J. Am. Chem. Soc.,111,1525-1527. 10. Teale, F. W. J., (1959), Biochim. Biophys. Acta, 35, 543-549. 11. Bradford M., (1976), Anal. Biochem., 72, 248-254. 12. Takano, T. J., (1977), J. Mol. Biol., 110, 537-568; Phillips, S. E. V., (1980), J. Mol. Biol., 142, 531-534. 13. Evans, S. V., Brayer, G. D., (1987), J. Biol. Chem., 263, 4263-4268. 14. Stetzkowski, F., Cassoly, R., Banerjee, R., (1979), J. Biol. Chem., 254, 1135111356. 15. Frydman, R. B., Bari, S., Tomaro, M. L. and Frydman, B., (1990), Biochem. Biophys. Res. Commun., 171,465-473.
1215
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ] 209-I 2"15
March 31, 1992
RECONSTITUTION OF APOMYOGLOBIN WITH EXTENDED BILIVERDINS
Marcelo Femfmdez, Rosalia B. Frydman, Sara Bad and Benjamin Frydman Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, Buenos Aires, Argentina
Received February 3, 1992
SUMMARY: An analysis of the reconstitution of biliverdins with extended conformations and horse heart apomyoglobin was carded out. Biliverdins with the 5Z-syn, lOZ-syn, 15Z-anti and 5Z-anti, lOZ-syn, 15Z-anti conformations, as well as biliverdins with the Z,Z,Z, all-syn conformation recombined with apomyoglobin. In every case the P enantiomers were bound in excess to the M enantiomers, with exception of the 5-syn, l O-syn,15-anti biliverdin where the M enantiomer bound preferentially to the protein. Biliverdins with an anti conformation at the C-10 meso bridge did not recombine with the protein. It was concluded that the presence of a syn conformation at the C-10 methine conferred to the biliverdin the necessary helicity to fit into the apomyoglobin heme pocket. This regioselectivity is of importance in view of the well known analogy between the ligand domains of myoglobin and the C-phycocyanins. ®1992 Academic Press,
Inc.
Bile pigments interact with proteins in two different ways. In the phycobiliproteins as well as in phytochrome they are covalently bound to the proteins by one or two thioether linkages and the protein enforces extended (or stretched) conformations on the bilitrienes as a result of electrostatic interactions between the propionate groups of the ligand and the basic residues of the proteins (1, 2). In the insect biliproteins known as insecticyanin (3) and biliverdin binding protein (BBP)(4), the biliverdin prosthetic groups are not covalently bound to the protein but interact with the latter through electrostatic and hydrophobic forces. In these biliproteins the biliverdins retain their energetically favored helical conformations. In the case of the phycobiliproteins, the biliverdins are derived from biliverdin IX t~ while in the insect biliproteins they are of the IX y type. It has been shown that apomyoglobin and the o~ and [3 subunits of C-phycocyanin share a similar "globin fold"; although their primary and secondary structures are quite different (5). This remarkable similarity led Falk and coworkers to analyze the recombination of sperm whale apomyoglobin with a number of helical biliverdins (6, 7). They found that apomyoglobin recombined with the biliverdins as long as the latter carded one or more propionate residues which allow them to anchor on the basic residues (His 97 and Arg 45) located at the entrance of the heme pocket. Their results led them to conclude that the apomyoglobin pocket is indifferent with respect to the steric and functional patterns present in the biliverdins (7). Since in the phycobiliproteins the biliverdins have extended conformations, recombination studies of apomyoglobin with extended
1209
0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
biliverdins are relevant to clarify if indeed steric factors are important for the interaction between the heme pocket of apomyoglobin and the biliverdins. An analysis of the binding of biliverdins with extended conformations to apomyoglobin should reveal to which extent the heme-binding domain of the latter and those present in the phycobiliproteins are analogous. We therefore explored the recombination of apomyoglobin and synthetic extended biliverdins with different degrees of stretching and with different configurations at the meso-bridges. The results showed that steric factors are crucial for the recombination of the biliverdins with the apomyoglobin and that the heme pocket in the latter is highly regioselective toward the conformation of the ligand.
MATERIALS AND METHODS Materials. Biliverdins IX ct l a , IX [3 l b , IX 7 lc and IX 8 l d (Fig.l) were obtained by the coupled oxidation of heme IX and were separated as described elsewhere (8). Biliverdins 2-6 (Fig. 1) were prepared by synthesis (9). Apomyoglobin was prepared by the acid-butanone method (10) from horse heart myoglobin (Sigma). All the reagents and solvents were of the highest analytical grade. PD-10 (Sephadex G-25) columns were from Pharmacia. Methods. In a typical preparation of an apomyoglobin-biliverdin complex, 15 mg of horse heart myoglobin were dissolved in 2 ml of 20 mM phosphate buffer, pH 6.0; the solution was adjusted to pH 2.8 with HC1, and the acid solution was extracted thrice with an equal volume of butanone. The berne-less apomyoglobin was dialyzed overnight against 20 mM phosphate buffer, pH 6.0. The apomyoglobin was then recombined with the biliverdins dissolved in a minimum volume of 0.1 N NaOH. The solution was adjusted to pH 6.0 with phosphate buffer. A ratio of 1:1.5 of protein to the chromophore was used. The mixture was allowed to stand for 2 h at 4 -0C and the
H
N--
R~
NH
N
,)7-NH
R4
N~-------~ 3_
Z
!
p
P~/N~ H
O.-
. ' ~ N ~ j ~ ,
p
£
5--~-X
5 P
l~r 0
_
6
~
_
N"
-0
P= CH2CH2CO2H
Figure 1. Structures of the biliverdins used in this study.la: biliverdin IX ¢t: Rl=R3=R6=R7=methyl; R4=R5=propionate; R2=R8=vinyl. l h : biliverdin IX ~: Rl=R4=R5=R7=methyl; R2=R3=propionate; R6=R8=vinyl. l c : biliverdin IX 3t: R2=R3=R5=R7=methyl; Rl=R8=propionate; R4=R6=vinyl. I t : biliverdin IX 8: Rl=R4=R6=R8=rnethyl; R2=R3=propionate; R5=R7=vinyl. 1210
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
complex was then isolated by filtration through a PD-10 column. The excess of biliverdin binds to the gel matrix and can be eluted with 0,1% Triton in the same buffer. When the dimethyl ester of biliverdin 2 was used for reconstitution, it was dissolved in the minimum volume of acetone so that upon mixing with apomyoglobin the final concentration of the acetone was less than 20%. After dialysis any excess of biliverdin ester was centrifuged off and the complex was filtered through the PD-10 column. Formation constants were calculated by quantitative interpretation of the visible difference spectra. In the case of biliverdin 2 we considered that e600 = e667 = 71.8 mM -1 cm -1. Protein concentrations were estimated by the method of Bradford (11) using bovine serum albumin as a standard. Optical spectra were measured with a double beam Hitachi U-2000 spectrophotometer. CD spectra were recorded with a JASCO J-20 Spectropolafimeter.
RESULTS Biliverdin IX o~ l a was recombined with horse heart apomyoglobin as described by Marko et al (6) for sperm whale apomyoglobin and essentially the same results were obtained. Horse heart and sperm whale myoglobin have identical heme cavities as revealed from their X-ray structures (11, 12) and from functional studies (13). The minor differences in the CD3 salt bridges of the apomyoglobin (Arg in sperm whale and Lys in horse heart myoglobin) should not affect their recombination properties with the biliverdins. The Cotton effect of the CD spectrum indicated that of the two helical enantiomers (P and M) of biliverdin IX c~ la, apomyoglobin binds preferentially to the P enantiomer, as reported by Marko et al. (6). Apomyoglobin was then recombined with biliverdins IX 13 lb, IX 7 lc and IX 8 ld. The UV/vis spectra of the complexes (Figs. 2A, 2C, and 2E) showed batochromic shifts in the short and long wavelength absorption bands. In the latter
I0
1
~7
\!
,,~l1-3~
A
670 l
B
=Lko
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t i
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0 05
I
I
-300
1-~75
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67O
0 C
3.0 _
_
J /
\
0
400
~-~
"-/ ~
s S°---
400
.
t 368
•
600
800
4(30
Sl~)
~
-IO.O
700
Wavelength, nm
Figure 3. (A): electronic absortion spectra of biliverdin 2 ( ) and biliverdin 2-apomyoglobin complex (----). (B): CD spectrum of the biliverdin 2- apomyoglobin complex. All the spectra were recorded in phosphate buffer, pH 6.0. 1212
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.J%
0.6
" l i
A
J
1
r
!
i
tlO
B
•
®
i
i
64o D
< ,
366
662
Ioo
D 357
-IO.O 200
400
600
800
400
500
660
[~ K)
Wavelength, nm
Figure 4. Electronic absortion spectra of biliverdin 3 (A), its dirnethyl ester (C) ( ), and of their complexes with apomyoglobin (----). The spectrum of biliverdin 3 dimethyl ester was recorded in acetone, while that of its complex with apomyoglobin was recorded in aqueous phosphate buffer, pH 6.0. CD spectra of biliverdin 3 (B) and biliverdin 3 dimethyl ester (D) apomyoglobin complexes.
the chromophores of the phycobiliproteins (1). Its visible absorption maxima suggests the presence of more than one conformational form, a fact that could result from different dihedral angle torsions at the C4-C5 and C9-C10 bonds. It recombines with apomyoglobin and the spectrum of the complex shows the usual batochromic shifts (Fig. 4 A, dashed line). The Cotton effect of its CD spectrum indicated a preferential binding of the M enantiomer (Fig. 4 B). It is the only biliverdin which has been recombined with apomyoglobin where a preferential binding of this enantiomer has been found. It stands in contrast with biliverdin IX 13where the P enantiomer binds preferentially (Fig. 2 B). Hence, the preference of the apomyoglobin pocket for the M enantiomer in 3 has to be attributed to the rigid anti configuration at C15. In the case of the dimethyl ester of 3 which also recombined with apomyoglobin (Fig. 4C) but only to ca. 15% of that of the free acid 3, the P enantiomer was the preferred one (Fig. 4 D). This fact underlines the great importance of the free propionate residues in the selective anchoring of one of the helical enantiomers in the myoglobin heme pocket. Biliverdins 4-6 (Fig. 1) did not recombine with apomyoglobin. This lack of recombination is noteworthy in biliverdin 4 where only the C10 double bond of the chromophore is part of an extended structure while the configurations at C5 and C15 keep the Z-syn chirality. The formation constants of the complexes of biliverdins IX (~ la, IX 13l b , IX y l e and IX 8 l d with apomyoglobin were found to be of the same order of magnitude (Table I). Among them the T isomer is the more readily complexed. In the case of biliverdin 2 (which is present in two conformations), two formation constants were calculated, one for each conformational form. The form absorbing at the longest wavelength had a formation constant thrice that of the other conformer (Table I). 1213
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Formation constants of the apomyoglobin-biliverdincomplexes Biliverdin
K ( M-1)
IX ~x la
3.4 x 105
IX [3 lb
1.6 x 105
IX y lc
8.0 x 105
IX8 ld
1.4x105
2 (617 nm)
4.0 x 105
2 (680 nm)
1.2 x 106
3
3.5 x 105
The constants were calculated as described in Materials and Methods.
DISCUSSION The obtained results confirm earlier data that the myoglobin heme-pocket is indifferent to the substitution type and pattern of the biliverdins (7). Changes in the former affect very little the complexation of the ligands with the protein. The results show, however, the critical importance of the steric factors for the complexation of the protein with the biliverdins. The lack of complexation of biliverdins 4-6 with apomyoglobin indicates the crucial importance of the configuration at the C10 bridge. When rings B and C are held in an extended conformation, no recombination with apomyoglobin takes place. Given the similarity of the "globin fold" of apomyoglobin and the phycocyanins, our results would explain why in the natural biliproteins of all types, the C10 bridge is always in the helically shaped
Z-syn configuration. Even the photodynamic equilibrium which
takes place in phytochrome affects only the isomerism at the C5 and C15 bridges (2). In the complexation of apomyoglobin with the four biliverdin IX isomers no major changes among them were found, either in the complexation constants or in the enantiomer discrimination. This suggests that the presence of biliverdin IX ot l a in the phycobiliproteins and of biliverdin IX y le in the insect biliproteins has a metabolic rather than a structural origin. A second factor which seems to be of relevance for the recombination of the biliverdins with apomyoglobin is the ability of the latter to preferentially bind one helical conformer. In every case when a biliverdin is bound to the apoprotein, a Cotton effect was detected in the CD spectra. In the more extended biliverdins (such as 4-6) no P/M isomerism is possible and this probably detracts from the flexibility needed by the ligand to fit into the protein pocket. The results reported above show how different the biliverdin binding domains of apomyoglobin (and therefore of phycocyanins) are from that of mammalian biliverdin reductase. The latter interacts with extended biliverdins to a much greater extent than with the helical ones (15). 1214
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS. This work was made possible by Grant GM-11973 from the National Institutes of Health (PHS). Support from CONICET (Argentina) is also acknowledged. We are indebted to Drs. H. Fern~indezand J.M. Delfino for recording the CD spectra. REFERENCES 1. Scheer, H. (1981) Angew. Chem. Int. Ed. Engl., 20, 241-261. 2. Riidiger, W. and Thiimmler, F. (1991) Angew. Chem. Int. Ed. Engl., 30, 1216-1228. 3. Riley, C. Y., Barbeau, B. K., Keim, P. S., Kezdy, F. J., Heinrickson, R. L. and Law, J. H. (1984) J. Biol. Chem., 259, 13159-13165. 4. Huber, R., Schneider, M., Mayr, I., MUller, R., Deutzmann, R.,Suter,F., Zuber H.,Falk, H. and Kayser, H. (1987), J. Mol. Biol., 198,499-513. 5. Schirmer, T., Bode, W., Huber, R., Sidler, W., and Zuber, H. (!985), J. Mol. Bio1.,184, 257-277. 6. Marko, H., MUller, N. and Falk, H. (1985), Monatsh. Chem., 120, 591-595. 7. Falk, H., Marko, H., MUller, N., Schmitzberger, W., and Stumpe, H. (1990), Monatsh. Chem., 121,893-901. 8. Frydman, R. B., Awruch, J. Tomaro, M. L., and Frydman, B. (1979),Biochem Biophys Res. Commun., 87, 928-935. 9. Iturraspe, J.B., Bari, S. and Frydman, B. (1989), J. Am. Chem. Soc.,111,1525-1527. 10. Teale, F. W. J., (1959), Biochim. Biophys. Acta, 35, 543-549. 11. Bradford M., (1976), Anal. Biochem., 72, 248-254. 12. Takano, T. J., (1977), J. Mol. Biol., 110, 537-568; Phillips, S. E. V., (1980), J. Mol. Biol., 142, 531-534. 13. Evans, S. V., Brayer, G. D., (1987), J. Biol. Chem., 263, 4263-4268. 14. Stetzkowski, F., Cassoly, R., Banerjee, R., (1979), J. Biol. Chem., 254, 1135111356. 15. Frydman, R. B., Bari, S., Tomaro, M. L. and Frydman, B., (1990), Biochem. Biophys. Res. Commun., 171,465-473.
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