Volulne 280, numt~r I. 199=201 FI~BS 09489 ~ 1~91 F~der~tlon of I~Atrol~¢,~tt~lily, heroical ~o¢I¢ti¢~tltl14~'/93~Jl~$l,~tl ADONI,~ 01114~79]~100199V
M,rch 1991
The reactivity of cytochrome c with soft ligands A b e l S c h e j t e r . B a t y a P l o t k i n a n d I d a Vig Lady Davix ('lmlr q f Bi¢~¢hcmislt;V. Sackler h~stilut¢ ¢~j'Mal¢¢ular Medicine. Soeklcr Mcttlcal Sch¢ml. Tel d vir UnivcrMO', 69978 Tel Aviv. hra¢l
Received 12 November 1990; rcvi~ed version received 13 J~muary 1991 The ~peetrul chanties caused by bindintt ~ort liLlnnd~tt~ the ¢yt,~¢itrt~me¢ iron =uld their ¢offehttit~nto lil~and allinitie~ ~uppt~rt the hypothe~i~that the iron-m~thionlne ~ulfi~r bond of thi~ heine protein i~ enhanced by deloeali~ation or the metal t.~t~le~tron~ into the empty 3d orhltnl~ of" the lisand aton~, The~e findins~ ~tlxoexplain the unique ~peetrtim or cyt(~d~r~me ¢ in tile fiw red. Cylochromc ¢; St~ft ligands; Iron-sulfur hond
1. I N T R O D U C T I O N An outstanding and hitherto unexplained characteristic o f c y t o c h r o m e c structure, is the ligation o f its iron by the sulfur of a methionine side-chain, a situation that is c o m m o n ~o all the k n o w n eukaryotic and prokaryotic c-type cytochromes [I]. Although thioether sulphur is a very p o o r ligand o f heine iron [2], the sulfur-iron bond o f cytochrome c is very strong, most esl~ecially in the reduced state of the metal [3]. A possible explanation o f this apparent a n o m a l y [4], based on the theory of hard and soft acids and bases [5], is that the cytochrome c low spin-iron 3d electrons delocalize into the orbitals o f w-acid or soft ligands, of which thioether sulfur is an example. T o test this hypothesis, the binding of the c y t o c h r o m e c iron with non-ionic ligands o f various d o n o r and acceptor a b i l i t i e s - thioether, phosphines, phosphite, lsocyanide and imidazole - was studied. This was facilitated by alkylation of the methionine-80 sulfur, which displaces it f r o m iron coordination [6] and leaves the metal accessible to exogenous ligands. Here we show that striking spectroscopic similarities exist between the native enzy me and its complexes with soft ligands, and that the stabilities of the reduced complexes are correlated to spectroscopic parameters indicative o f the extent of back-bonding in these complexes. 2. M A T E R I A L S A N D M E T H O D S Horse heart cytochron~e c, type 111 (Sigma Cl~emical Co., St. Louis), was purified [7] and modified into dicarbo×ymethyl cytochrome c (di-CM-c) [6]. Tributyl phosphine (TBP), Correspo~tdence address: A. Schejter, Sacklcr Institute of Molecular Medicine Sackler Medical School, Tel Aviv University, 69978 Tel Arty, Israel
Published by Elsevier Science Publishers B. V.
dimethylphenyl pho$1}hitle (DMPP), trimethyl pllo~phite (TMP), dimethyl .~ulfide (DMS), imidazole (Ira) and butyl i~oeyanide (BIC) ~ere fram Aldrich Chemical Co.. Milwaukee. Optical spectra were recorded on Cary 14 an0 Cary 219 instruments. 3. R E S U L T S C o m p l e x e s of reduced and oxidized di-CM-c were o b t a i n e d by adding T B P in ethanolic solution. For ferrous di-CM-c, the visible b a n d s shifted f r o m 550 n m and 520 nm, to new peaks at 557 n m and 528 nm (Fig. 1). T h e ~ band Lost intensity relative to tile ~ b a n d (Table I). In the far red, a weak but sharp band at 688 nm and two additional b r o a d bands centered at 760 and 810 n m , were observed (Fig. 2). Similar bands were present in the complexes of ferrous di-CM-c with D M P P , T M P , Ira, BIC and DMS, whose spectroscopic features in the visible and Soret regions are listed in Table I. Except for imidazole, all the ferric complexes had weak b a n d s in the far red, where c y t o c h r o m e c has its characteristic 695 nrn band [8,9]. T h e spectra with best resolved bands are presented in Fig. 3. Titrations of the binding o f ligands by ferrous diC M - c at various p H s showed Hill plots with slopes o f 0.9-1.1; binding constants with the u n p r o t o n a t e d ligands, are shown in Table 1. 4. D I S C U S S I O N Trivalent phosphorus c o m p o u n d s are very g o o d ligands because they are strong o-donors [10]; they are also g o o d ~r-acceptors because o f their empty, low-lying 3d orbitals, a p r o p e r t y t h a t they share with divalent sulfur ligands; BIC has molecular orbitals with strong a c c e p t o r properties; imidazole is a far stronger base than DMS, BIC or T M P , but a m u c h poorer ~r-acid. If these ligands are a r r a n g e d in terms of their decreasing
199
March 1991
FEB$ L E'I'TE~
Volum~ 21t0, number 2
!
e
~o
~o
°
a.d
I. complex with trlbu|yl phosphh~¢ ( ...... ),
affinities for ferrous di-CM-c, tl~e following series is obtained: TMP > D M P P >BIC, TBP >DMS >Ira. The strongest affinities are those of the strongest racceptors, indicating that the softness of the ligands [5] plays an important role in determining their affinities for the di-CM-c iron. The spectral effects of the ligands on the energies and intensities of the visible bands are roughly correlated: ligands that shift the bands to the red, decrease the ratio of intensities of t h e n and B bands. Aligning the ligands according to the decreasing energies of the baricenters of their o and Soret bands
650
750
8,60
WAVELENGTH
[ nm
Fig. 2. Optical absorption spectra of ferrous di.CM.¢ ( - - ) and its complex with tributyl phosphine ( ...... ) in the far red,
[111, the following order is obtained: T B P < D M P P < T M P < BIC >DMS < Ira. Thus, soft ligands have the bathochromic effects on the porphyrin bands o f di-CMc that should be expected from metal electron Table I
l..igand
None
Absorption maxima (nm)
Binding constants
(AbsorbanCies ( r a M " ~ • c" ~))
(I M - t)
414
520
549
(~ 17)
0 1,8)
(22,3)
[0.761
[0,075]
[0.0481
BIC
423 (151) [0.671
522 (14.9) [0.075]
552 (18,9) [0.034]
2.5 × 104
h'n
414 (112) [0,66]
520 (13,5) [0.064]
549 (28,t) [0.042]
8.7 x 102
TBP
426
528
557
2.2 × 104
(118)
(15.8)
(21.3)
E0.65]
[0.081 ]
[0,032]
DMPP
425 (97)
528 (12,6)
557 (14,6)
[0.7 I1
[0.072]
[0.0191
TMP
424 (109) [0.701
523 (13.2) [0.067]
552 (I7,1) [0,034]
8.3 x 104
DMS
416 (123) [0.62]
522 (14,8) [0,06~]
551 (25.8) ~0,043]
2.5 × 103
200
$.0 x 104
Volume ;11f0, number 2
FE'BS L E T T E R S
March 199I
cytochromc c in the far red, the spectral reBion in which tile d..d and porphyrin ~r-d transitions are observed. These char/3e transfer and d-d bands, uniquely characteristic of ferrous cytochrome c, where they ap. pear between 600 and 900 nm [I5l, are reproduced by tl~e ferrous di.CM-c complexes with TBP. In the ferric state ttle spectrum of cytochrome c is outstanding
i II ql
|
s @el@ @e@ o@ ~@ @ i 0 g20
6e0
740 WAV|LINGTH
~
I~00
I nml
Fit. 3, Optical absorption spectra or ferric di.CM-e complexeswith butyl isocyanide (--), dimethyl sulfide ( ...... ] and trimethyl phosphite (...... ) in the far red. dclocalization into their orbitals [12,13]. A similar series, D M P P < BIC < T M P < T B P < DMS < Ira, is obtained when the ligands are o r d e r e d according to the decreasing ratios o f t h e intensities o f the o~ a n d / ~ b a n d s , ~,,/~j, as is typically found in low-spin f e r r o u s home complexes with ligands o f increasing ~r-accepting p o w e r [ l l ] ; or when they are ordered according to the ratios of the oscillator strengths o f the same bands (Table I), (Oscillator strength = 4.6 x 1 0 " ~ . e,..~ , Atn, where e,...~ is the m o l a r absorptivity of the band, and .x,/a its width in c - n where its absorptivity has half o f its maximal value [14].) The similarities between the three series o f ligands, two defined by spectroscopic correlates o f b a c k - b o n d i n g and the other by affinities to ferrous di-CM-c, is in keeping with the hypothesis that the c y t o c h r o m e c ~ron has strong covalent tendencies [4]. The hypothesis is also supported by the spectral similarities between the complexes of d i - C M - c with soft ligands and native
because o f the presence o f a well defined band at 695 nm ['/,8]. Th~ o b s e r v a t i o n of corresponding bands in the complexe~ o f d i . C M - c with. soft ligands (Fig, 3) suggests that t h e electro=tic c o n f i g u r a t i o n s of" the iron ~nd its ligand field in these complexes are very similar to that o f native c y t o c h r o m e c, and explains the unique o b s e r v a t i o n of these b a n d s in c y t o c h r o m e c and s o m e o f its complexes as due to a combinatLon o f two factors in the sixth iron ligand: a weak or moderate ligand field, and a strong or m o d e r a t e rr-accepting power. REFERENCES
[I] Matthews, F,S. (1985)Prog, Biophys, Mol. Biol. 45. !-56, [2] Ellis, P,E,. Jones, R.D. and Basolo, F. (1979) Proc, Nell, Acad. Sci. U S A 96, 5418-5420, [3] Margoliash, E, and Schejter, A, (1966) Adv. Protein Chem, 21, I 13-283.
(4] Schejter, A. and Plotkin, B, (1988) Biochem, J. 255,353-356. [S] Pearson, R,G, (1963) J. A m . Chem. Soc 85 353 ~ s539. [6] Schejter, A, and Avirarn, [, (1970) J. Bic.~. Chem. 295,
1552-1557, [7] Margoliash, E. and Walassek, O,F, (1967) Methods Enzymol. 10. 339-348. [8] Schejter, A, and George, P, (1964) Biochemistry 3, I045-I049,
[9] Eaton, W.A and Hochstrasset, R, (196'7) J, Chem. Phys. 46, 2533-2539 [10] Streuli, C.A, (1960) Anal. Chem, 32, 985-987. ill] Hill, H.O.A., Roder, A; and Williams, R.J,P, (1970)Struet. Bond. 8, 123-151, [12] Gouterman, M, (1978) in: The Porphyrins. vol 3 (Dolphin, D. ed,), pp. 1-165, Academic Press, New York [13] Antipas, A,, Buchler, J.W., Gouterman, M. and Smith, P.D,, J, Am, Chen~. Soe. [00, 3015-3030, [14] Lever, A,B,P, (I 984) Inorganic Electronic Spectroscopy (2nd ed) Elsevier. Amsterdam, [15] Eaton, W,A and Charney, E. (1969) J, Chem. Phys, 50, 4502-4505,
201
M,rch 1991
The reactivity of cytochrome c with soft ligands A b e l S c h e j t e r . B a t y a P l o t k i n a n d I d a Vig Lady Davix ('lmlr q f Bi¢~¢hcmislt;V. Sackler h~stilut¢ ¢~j'Mal¢¢ular Medicine. Soeklcr Mcttlcal Sch¢ml. Tel d vir UnivcrMO', 69978 Tel Aviv. hra¢l
Received 12 November 1990; rcvi~ed version received 13 J~muary 1991 The ~peetrul chanties caused by bindintt ~ort liLlnnd~tt~ the ¢yt,~¢itrt~me¢ iron =uld their ¢offehttit~nto lil~and allinitie~ ~uppt~rt the hypothe~i~that the iron-m~thionlne ~ulfi~r bond of thi~ heine protein i~ enhanced by deloeali~ation or the metal t.~t~le~tron~ into the empty 3d orhltnl~ of" the lisand aton~, The~e findins~ ~tlxoexplain the unique ~peetrtim or cyt(~d~r~me ¢ in tile fiw red. Cylochromc ¢; St~ft ligands; Iron-sulfur hond
1. I N T R O D U C T I O N An outstanding and hitherto unexplained characteristic o f c y t o c h r o m e c structure, is the ligation o f its iron by the sulfur of a methionine side-chain, a situation that is c o m m o n ~o all the k n o w n eukaryotic and prokaryotic c-type cytochromes [I]. Although thioether sulphur is a very p o o r ligand o f heine iron [2], the sulfur-iron bond o f cytochrome c is very strong, most esl~ecially in the reduced state of the metal [3]. A possible explanation o f this apparent a n o m a l y [4], based on the theory of hard and soft acids and bases [5], is that the cytochrome c low spin-iron 3d electrons delocalize into the orbitals o f w-acid or soft ligands, of which thioether sulfur is an example. T o test this hypothesis, the binding of the c y t o c h r o m e c iron with non-ionic ligands o f various d o n o r and acceptor a b i l i t i e s - thioether, phosphines, phosphite, lsocyanide and imidazole - was studied. This was facilitated by alkylation of the methionine-80 sulfur, which displaces it f r o m iron coordination [6] and leaves the metal accessible to exogenous ligands. Here we show that striking spectroscopic similarities exist between the native enzy me and its complexes with soft ligands, and that the stabilities of the reduced complexes are correlated to spectroscopic parameters indicative o f the extent of back-bonding in these complexes. 2. M A T E R I A L S A N D M E T H O D S Horse heart cytochron~e c, type 111 (Sigma Cl~emical Co., St. Louis), was purified [7] and modified into dicarbo×ymethyl cytochrome c (di-CM-c) [6]. Tributyl phosphine (TBP), Correspo~tdence address: A. Schejter, Sacklcr Institute of Molecular Medicine Sackler Medical School, Tel Aviv University, 69978 Tel Arty, Israel
Published by Elsevier Science Publishers B. V.
dimethylphenyl pho$1}hitle (DMPP), trimethyl pllo~phite (TMP), dimethyl .~ulfide (DMS), imidazole (Ira) and butyl i~oeyanide (BIC) ~ere fram Aldrich Chemical Co.. Milwaukee. Optical spectra were recorded on Cary 14 an0 Cary 219 instruments. 3. R E S U L T S C o m p l e x e s of reduced and oxidized di-CM-c were o b t a i n e d by adding T B P in ethanolic solution. For ferrous di-CM-c, the visible b a n d s shifted f r o m 550 n m and 520 nm, to new peaks at 557 n m and 528 nm (Fig. 1). T h e ~ band Lost intensity relative to tile ~ b a n d (Table I). In the far red, a weak but sharp band at 688 nm and two additional b r o a d bands centered at 760 and 810 n m , were observed (Fig. 2). Similar bands were present in the complexes of ferrous di-CM-c with D M P P , T M P , Ira, BIC and DMS, whose spectroscopic features in the visible and Soret regions are listed in Table I. Except for imidazole, all the ferric complexes had weak b a n d s in the far red, where c y t o c h r o m e c has its characteristic 695 nrn band [8,9]. T h e spectra with best resolved bands are presented in Fig. 3. Titrations of the binding o f ligands by ferrous diC M - c at various p H s showed Hill plots with slopes o f 0.9-1.1; binding constants with the u n p r o t o n a t e d ligands, are shown in Table 1. 4. D I S C U S S I O N Trivalent phosphorus c o m p o u n d s are very g o o d ligands because they are strong o-donors [10]; they are also g o o d ~r-acceptors because o f their empty, low-lying 3d orbitals, a p r o p e r t y t h a t they share with divalent sulfur ligands; BIC has molecular orbitals with strong a c c e p t o r properties; imidazole is a far stronger base than DMS, BIC or T M P , but a m u c h poorer ~r-acid. If these ligands are a r r a n g e d in terms of their decreasing
199
March 1991
FEB$ L E'I'TE~
Volum~ 21t0, number 2
!
e
~o
~o
°
a.d
I. complex with trlbu|yl phosphh~¢ ( ...... ),
affinities for ferrous di-CM-c, tl~e following series is obtained: TMP > D M P P >BIC, TBP >DMS >Ira. The strongest affinities are those of the strongest racceptors, indicating that the softness of the ligands [5] plays an important role in determining their affinities for the di-CM-c iron. The spectral effects of the ligands on the energies and intensities of the visible bands are roughly correlated: ligands that shift the bands to the red, decrease the ratio of intensities of t h e n and B bands. Aligning the ligands according to the decreasing energies of the baricenters of their o and Soret bands
650
750
8,60
WAVELENGTH
[ nm
Fig. 2. Optical absorption spectra of ferrous di.CM.¢ ( - - ) and its complex with tributyl phosphine ( ...... ) in the far red,
[111, the following order is obtained: T B P < D M P P < T M P < BIC >DMS < Ira. Thus, soft ligands have the bathochromic effects on the porphyrin bands o f di-CMc that should be expected from metal electron Table I
l..igand
None
Absorption maxima (nm)
Binding constants
(AbsorbanCies ( r a M " ~ • c" ~))
(I M - t)
414
520
549
(~ 17)
0 1,8)
(22,3)
[0.761
[0,075]
[0.0481
BIC
423 (151) [0.671
522 (14.9) [0.075]
552 (18,9) [0.034]
2.5 × 104
h'n
414 (112) [0,66]
520 (13,5) [0.064]
549 (28,t) [0.042]
8.7 x 102
TBP
426
528
557
2.2 × 104
(118)
(15.8)
(21.3)
E0.65]
[0.081 ]
[0,032]
DMPP
425 (97)
528 (12,6)
557 (14,6)
[0.7 I1
[0.072]
[0.0191
TMP
424 (109) [0.701
523 (13.2) [0.067]
552 (I7,1) [0,034]
8.3 x 104
DMS
416 (123) [0.62]
522 (14,8) [0,06~]
551 (25.8) ~0,043]
2.5 × 103
200
$.0 x 104
Volume ;11f0, number 2
FE'BS L E T T E R S
March 199I
cytochromc c in the far red, the spectral reBion in which tile d..d and porphyrin ~r-d transitions are observed. These char/3e transfer and d-d bands, uniquely characteristic of ferrous cytochrome c, where they ap. pear between 600 and 900 nm [I5l, are reproduced by tl~e ferrous di.CM-c complexes with TBP. In the ferric state ttle spectrum of cytochrome c is outstanding
i II ql
|
s @el@ @e@ o@ ~@ @ i 0 g20
6e0
740 WAV|LINGTH
~
I~00
I nml
Fit. 3, Optical absorption spectra or ferric di.CM-e complexeswith butyl isocyanide (--), dimethyl sulfide ( ...... ] and trimethyl phosphite (...... ) in the far red. dclocalization into their orbitals [12,13]. A similar series, D M P P < BIC < T M P < T B P < DMS < Ira, is obtained when the ligands are o r d e r e d according to the decreasing ratios o f t h e intensities o f the o~ a n d / ~ b a n d s , ~,,/~j, as is typically found in low-spin f e r r o u s home complexes with ligands o f increasing ~r-accepting p o w e r [ l l ] ; or when they are ordered according to the ratios of the oscillator strengths o f the same bands (Table I), (Oscillator strength = 4.6 x 1 0 " ~ . e,..~ , Atn, where e,...~ is the m o l a r absorptivity of the band, and .x,/a its width in c - n where its absorptivity has half o f its maximal value [14].) The similarities between the three series o f ligands, two defined by spectroscopic correlates o f b a c k - b o n d i n g and the other by affinities to ferrous di-CM-c, is in keeping with the hypothesis that the c y t o c h r o m e c ~ron has strong covalent tendencies [4]. The hypothesis is also supported by the spectral similarities between the complexes of d i - C M - c with soft ligands and native
because o f the presence o f a well defined band at 695 nm ['/,8]. Th~ o b s e r v a t i o n of corresponding bands in the complexe~ o f d i . C M - c with. soft ligands (Fig, 3) suggests that t h e electro=tic c o n f i g u r a t i o n s of" the iron ~nd its ligand field in these complexes are very similar to that o f native c y t o c h r o m e c, and explains the unique o b s e r v a t i o n of these b a n d s in c y t o c h r o m e c and s o m e o f its complexes as due to a combinatLon o f two factors in the sixth iron ligand: a weak or moderate ligand field, and a strong or m o d e r a t e rr-accepting power. REFERENCES
[I] Matthews, F,S. (1985)Prog, Biophys, Mol. Biol. 45. !-56, [2] Ellis, P,E,. Jones, R.D. and Basolo, F. (1979) Proc, Nell, Acad. Sci. U S A 96, 5418-5420, [3] Margoliash, E, and Schejter, A, (1966) Adv. Protein Chem, 21, I 13-283.
(4] Schejter, A. and Plotkin, B, (1988) Biochem, J. 255,353-356. [S] Pearson, R,G, (1963) J. A m . Chem. Soc 85 353 ~ s539. [6] Schejter, A, and Avirarn, [, (1970) J. Bic.~. Chem. 295,
1552-1557, [7] Margoliash, E. and Walassek, O,F, (1967) Methods Enzymol. 10. 339-348. [8] Schejter, A, and George, P, (1964) Biochemistry 3, I045-I049,
[9] Eaton, W.A and Hochstrasset, R, (196'7) J, Chem. Phys. 46, 2533-2539 [10] Streuli, C.A, (1960) Anal. Chem, 32, 985-987. ill] Hill, H.O.A., Roder, A; and Williams, R.J,P, (1970)Struet. Bond. 8, 123-151, [12] Gouterman, M, (1978) in: The Porphyrins. vol 3 (Dolphin, D. ed,), pp. 1-165, Academic Press, New York [13] Antipas, A,, Buchler, J.W., Gouterman, M. and Smith, P.D,, J, Am, Chen~. Soe. [00, 3015-3030, [14] Lever, A,B,P, (I 984) Inorganic Electronic Spectroscopy (2nd ed) Elsevier. Amsterdam, [15] Eaton, W,A and Charney, E. (1969) J, Chem. Phys, 50, 4502-4505,
201
