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Characterization of three iron ferredoxins by microwave power saturation
BIOCItlM~E, 1984 66~ 77-79
Characterization of three iron ferredoxix~s by microwave power saturation. Patrick BERTRAND*, Bruno GUIGLtARELLI*. Jacques MEYER**, and Jean-Pierre GAYDA*. * Laboratoire d'Eiectronique des MiJieux Condensds (E.R.A. 375/, Universitd de Frovence, 13397 Mar~eille Cedex 13 {France), ** Labaratoire de Biochimie, D.R.E - C.E.N.G., Gre~oble (France/. (Regu le 7-I2-1983. accept? le 4-1-]9~'4:
R~sum4 Nous aeons mesurd /e temps de relaxation dlecgromque spin-rdseau T~ dans la gamme de tempdrature 4 K-IO K, par saturation continue du signal R.£E. des ferredoxines a 3 fer de Desulfovibrio glgas et Azotobacter vine!andii. La compamison des r&uYars avec crux obtenus precddemmem sur d'autres O'pes de prntdines fer-souj?e', met en d~,idence les propridtds de relaxation partieulio2remem rapide du signal R.P.E. dans les j?rredoxines h trois jot. Ces ~dsulmts sont en aceorJ aver !es mod?les de centre aetif qui prfvoient Fe::'is~_encede ~Tiveaux excit& frOs proches. -
Mots-el~
-
: pruC/~irie fer-suufre / temps de reiuxat~or~ / R.P.E~
S u m m a r y - - We have measured the electronic spin lattice re]devotion tinge T, in the iengperature range 4 K-IO g by .micro~vaw',po~'er saturation on the 3Fe ferredo_:~insfrom Desulfovibrio gigas and ~2otobacter vine!andii. ;r/Te comparison with the resu#s previously obtained on other iron sulfur proteins emphasizes the ~rticutartv fast relaxing properties of the E.P.R. signal in 3Fe ferredoxins. These results support the models oj.~he active s:ge ~:"~ichpred)'c," veO, low (v~ng excited levels. Key-words : iro~ sulfur proteiv., / retaxet~m~time / E.P.R.
In 1980, the discovery of 3Fe type ferredcxins raised a considerable interest in the field of iron sulfur proteins research. Since this date, the number of proteins which are believed to belong to this new class is rapidly growing Ill. In ~he oxidized state of the protein, the three Fe(HI) atoms are coupled by strong antfferromagnetic exchange interactions, This leads to a S = 1/2 gronnd ~-~ate which gives a peculiar nearly isotropic E.P.R. spectrum in the g > 2 range (fig. 1). The temperature dependence of this spectrum
appears very characteristic, with a modification of the low t~e!d part between 8 K and 16 K, and an overact broader~ing above 16 K (fig. l), This broadening is due to the shor~ening of the electronic spin-lattice relaxation time T . We showed previously how the study of this temperature dependence could bring information about the nature dud the energy of the lowest excited leveis in the 3Fe clusters [2, 3}. In this work, we present an extension of T: measurements to the temperatare range 4 K -- i0 K, obtained by microwave power saturation on the 3Fe ferredoxi~'~s from Desulfovibrio
P. Bertrand a n d ceq.
78
D,g. Fd :~Y
~ A.v. Fd T
g=2.007
!tl' g=2,0~2
ff
Science, Kings College, London. It was prepared according to the method described in [5]. The spectra were ob~:aiped on a VARIAN Ell2 E.P.R. spectrometer. The samples were cooled whh a gas flow Air Prcducts System. The temperature was measured before and a!'ter each spectrum with a calibrated thermocouple (ch~t, ine! v S Au -- 0.07 per cent Fe) placed in an E.P.R. tube partially filled with water. In the continuous sat=.ration experiments_ we used the method reported in [6] to extend Casmer's model [7]. For the three samples studied in these experiments (Dg. Fd tI, ,q.v. Fd l, chromatium HIPIP), T~ was measured on the high field peak of the E.P.R, spectrum. By simulating the satmation of calculated spectra, v,e checked that the saturation curves of these very inhomogeneous derivativeqi:,= peaks were not sensitive to their detailed shape, at .e:,st in the range of available saturating powers. 2~:; ,~nab'cd : : tc use .uc mcthJ,i prcsent~_~ i;; [~1, which applied originally to gaussian absorption-like peaks. in the relaxation broadening experiments performed on the HIPIP sample, we used the method reported in [8] to measure T oil the high field part of the spectrum.
ResMts FIG 1. Teinptratltre dependetlcc l!f t/te' E [~R. spet'ttzl uf nro 3Fe f~'rredorin.~. Desulfovibno gigas [ertrdo~in ]I zD.g. Fd [l) and Azotobacter ~inelandi: ferredori!l ! ,A.~ Fd !~ The concentration of the Iwo samples are about 2 mM and 1 mM. respectively. Experimental conditions : modulatic,n 100kHz, 2Gauss p.p. mierowa,,e power from 0I mW t¢~ I roW,
gigas and A z o m b a c t e r vinelandii. As a comparison, we also p e r f o r m e d similar m e a s u r e m e n t s on the H I P I P f r o m C h r g m a t i u m vh~osum, tn its oxidized state, this [4Fe 4S] protein also gives a weakly anisotropic E.P.R. spectrum in the g ~> 2 range, which was sometimes confused with 3Fe clusters signals.
The results o f this study e m p h a s i z e the particularly fast rela~zing properties o f the E . P R . signals in 3Fe f e r r e d e d n s .
Materials and methods ,'Pae D. gigas ferredoxin il (D.g. Fd II) sample is described in reference [2]. A. vmelandii ferredoxin I (A.v. Fd I) was purified according to the method described in [4]. The Chromatium HIPIP sample was a gift from Dr. R. Cammack, Department of Plant
They are presented on figure 2. The similarity o f the T~ values m e a s u r e d at high temperature on D.g. Fd It a n d A.v. Fd I [3] is also apparent ;n the low temperature range. They are much s l e r than those previously obtained on [2Fe-2S] [6] and [4Fe-4S] clusters [9]. It is worthwfle to c o m p a r e these properties with the relaxation b e h a v i o u r o f the [4Fe-4S] H I P I P from chromatium, which also gives a weakly anisotropic signal in the g >~ 2 ,ange in its oxidized state. The values o f T~ m e a s u r e d by continuous saturation on this s a m p l e , are in good a g r e e m e n t with the values which can be obtained from the saturation curves published in [5] (fig. 2). H o w e v e r the relaxation times T~ derived f r o m the spectral b r o a d e n i n g are significantly longer than those reporied by Blum et aL [10]. In fact these authors m e a s u r e d the b r o a d e n i n g of integrated spectra, whereas we studied the b r o a d e n i n g o f the high field part o f the derivative spectrum. Nevertheless, a careful e x a m i n a t i o n of their data reveals that the discrepancy cannot be attributed to the different methods, but is already included in the experimental results. In particular, their published spectra a p p e a r m u c h more b r o a d e n e d than those we recorded at the s a m e temperature. This c o m p a r i s o n shows that for T >~ 10 K, the
C/laraeterizat on o/' l!~ree !~'on /errede~:ins by microw~ave D .¢er saturation
k (s -! )
d~ :~'/i" •"-/' / / ". ""
109
, . , ,g ° /
10 ) !
,'
/a
At low temperature, the relaxation o f the HI PIP from C h r o m a t i u m aiso appears very efiiciem (fig. 2). As in the ,ease o f 3Fe ;'erredoxins. this ~ould resttlt from the remova! of ~he S = I / 2 degeneracy.
105
I
1
/
[
,
105 I
r
~nis explanation is compatible ,*i,'h the tempecature depender~ce o( the magnetic susceptibihty in this protelrL which can be interpreted by an Fieisenberg hamittonian ~ h e r e the difference between the J va!ues can reach l0 e r cent [1ti.
.~[/
,ol
T(K)
t ~'~(, 2
79
range of temFe<~,q~re where their E.P.R, signal can he o b s e r v e d ~his properly is certain{y related to the presence o f several low lyir~g excited states (cf. t]g. 5 o f ref. 2). |n particular, the reiaxation broadening observed for temperatures higher than 20 K i-: probably determined by the S = 3/2 states whose energy is about 50-100cm ~, whereas the ve*'y efficient telaxation below t 0 K could result trove the existence of the first S = 1 2 e~cited level ~;hose energy is only about 2*; cm : [2, 31.
2
5
10
-- [~-mpi'ramre depct~dcI!~
20
50
REFERENCES
o/ tkl ~/ava:t,,~ t m ~
1 Beinen, H g: Thorn,ion. A.J. (19t~3; 5ullur l~r,Jh'ttt~
SFe ferre&,~ins , f)~ [-d It. e 4 ~ f-d ; [4Fe-4Sl HIPIP from (hr, m*u:tlm ~m.~um . ,~k*r me:~,urcments. ,: T cadctztalcd Ire m ~h~ ~au~ ~tion cur~e:, published in vet [5}. from rot It~) ~ther irwin ,ulfur pr,:,ieiz~s .-- & {2Fc-251 {ccr~-d,~xz~ [~t,m :;p~rMina ma~tm,i [6~ -- B {~[e~tS~ fc~re,Jcxm fr:.r~ ~alll/ti~ Ml'li~¢lIil,rme)pIilh;I [91
spin-lattice relaxation in the 3Fe proteins D.g. Fd H and A.v. Fd ! is much more efficient thar~ in the other class o f iron sulfur proteins, including ~he HIP1P from C h r o m a t i u m
Discussion This study demonstrates that the 3Fe 7"erredoxins are vet 3 fast relaxing centers in the whole
Arch. ,~iochem
Biophya, 2,22, 333461. 2. Gayda, J.P., Be~rznd, P. & Th~aodu!e, F.X. (i9821 J. Chem. Ph)w., "t7. 3387-3391. 3 Ga}da. J.P., Bertrand. P.. Gaigliarelli. B. & Meyer. J. {1983~ J Chem. Phil, 79, 5732-5733. 4. Ghe,~b. D. O'DoqnelL S.. Furey. W.J.R.. Robbins. A.H. & Stout, C,D, (t982; Z MoL BioL ~58, 73. 5. Rupp. H., Rao, K.K.. D,O. & Cammack, R. (1978) B]o.him Biophy,. 4c~a. 33"/, 255-269. ~-. Gayda, J,P. Bertrand, P, De~i]le. A., More, C., Roger, G., Gibson. J-F. & Cammack. R, (]979~ [3iocbtm Biophvs. Ac~a. $1gI, 15-26. 7, Casmer, T.O. (19591 Phys, Rev.. t~5, 1506-15t5, 8. Berwan& P, Roger, G. & Gayda, J.P. t1980) ,L Magn. Res.. 4{~, 53g'-549 9. ~e~rand. P., Gasda. J P. & Rao. K.K. (i982) Z ('hem. ph, vs.. °%, 4715-4"719. i 0 g~un~, H., Sa{emo. JC., Prithee. R.C., Leig=h, 3.S. & Obnish{, 1-. U977) Bk~ph)'s .L. 20, 23-3?. il Anlanai~is. B.C & Mo~s, T.H. (1975) Bi6,chim. Biuph:,s Arm. 405, 262-279.
Characterization of three iron ferredoxix~s by microwave power saturation. Patrick BERTRAND*, Bruno GUIGLtARELLI*. Jacques MEYER**, and Jean-Pierre GAYDA*. * Laboratoire d'Eiectronique des MiJieux Condensds (E.R.A. 375/, Universitd de Frovence, 13397 Mar~eille Cedex 13 {France), ** Labaratoire de Biochimie, D.R.E - C.E.N.G., Gre~oble (France/. (Regu le 7-I2-1983. accept? le 4-1-]9~'4:
R~sum4 Nous aeons mesurd /e temps de relaxation dlecgromque spin-rdseau T~ dans la gamme de tempdrature 4 K-IO K, par saturation continue du signal R.£E. des ferredoxines a 3 fer de Desulfovibrio glgas et Azotobacter vine!andii. La compamison des r&uYars avec crux obtenus precddemmem sur d'autres O'pes de prntdines fer-souj?e', met en d~,idence les propridtds de relaxation partieulio2remem rapide du signal R.P.E. dans les j?rredoxines h trois jot. Ces ~dsulmts sont en aceorJ aver !es mod?les de centre aetif qui prfvoient Fe::'is~_encede ~Tiveaux excit& frOs proches. -
Mots-el~
-
: pruC/~irie fer-suufre / temps de reiuxat~or~ / R.P.E~
S u m m a r y - - We have measured the electronic spin lattice re]devotion tinge T, in the iengperature range 4 K-IO g by .micro~vaw',po~'er saturation on the 3Fe ferredo_:~insfrom Desulfovibrio gigas and ~2otobacter vine!andii. ;r/Te comparison with the resu#s previously obtained on other iron sulfur proteins emphasizes the ~rticutartv fast relaxing properties of the E.P.R. signal in 3Fe ferredoxins. These results support the models oj.~he active s:ge ~:"~ichpred)'c," veO, low (v~ng excited levels. Key-words : iro~ sulfur proteiv., / retaxet~m~time / E.P.R.
In 1980, the discovery of 3Fe type ferredcxins raised a considerable interest in the field of iron sulfur proteins research. Since this date, the number of proteins which are believed to belong to this new class is rapidly growing Ill. In ~he oxidized state of the protein, the three Fe(HI) atoms are coupled by strong antfferromagnetic exchange interactions, This leads to a S = 1/2 gronnd ~-~ate which gives a peculiar nearly isotropic E.P.R. spectrum in the g > 2 range (fig. 1). The temperature dependence of this spectrum
appears very characteristic, with a modification of the low t~e!d part between 8 K and 16 K, and an overact broader~ing above 16 K (fig. l), This broadening is due to the shor~ening of the electronic spin-lattice relaxation time T . We showed previously how the study of this temperature dependence could bring information about the nature dud the energy of the lowest excited leveis in the 3Fe clusters [2, 3}. In this work, we present an extension of T: measurements to the temperatare range 4 K -- i0 K, obtained by microwave power saturation on the 3Fe ferredoxi~'~s from Desulfovibrio
P. Bertrand a n d ceq.
78
D,g. Fd :~Y
~ A.v. Fd T
g=2.007
!tl' g=2,0~2
ff
Science, Kings College, London. It was prepared according to the method described in [5]. The spectra were ob~:aiped on a VARIAN Ell2 E.P.R. spectrometer. The samples were cooled whh a gas flow Air Prcducts System. The temperature was measured before and a!'ter each spectrum with a calibrated thermocouple (ch~t, ine! v S Au -- 0.07 per cent Fe) placed in an E.P.R. tube partially filled with water. In the continuous sat=.ration experiments_ we used the method reported in [6] to extend Casmer's model [7]. For the three samples studied in these experiments (Dg. Fd tI, ,q.v. Fd l, chromatium HIPIP), T~ was measured on the high field peak of the E.P.R, spectrum. By simulating the satmation of calculated spectra, v,e checked that the saturation curves of these very inhomogeneous derivativeqi:,= peaks were not sensitive to their detailed shape, at .e:,st in the range of available saturating powers. 2~:; ,~nab'cd : : tc use .uc mcthJ,i prcsent~_~ i;; [~1, which applied originally to gaussian absorption-like peaks. in the relaxation broadening experiments performed on the HIPIP sample, we used the method reported in [8] to measure T oil the high field part of the spectrum.
ResMts FIG 1. Teinptratltre dependetlcc l!f t/te' E [~R. spet'ttzl uf nro 3Fe f~'rredorin.~. Desulfovibno gigas [ertrdo~in ]I zD.g. Fd [l) and Azotobacter ~inelandi: ferredori!l ! ,A.~ Fd !~ The concentration of the Iwo samples are about 2 mM and 1 mM. respectively. Experimental conditions : modulatic,n 100kHz, 2Gauss p.p. mierowa,,e power from 0I mW t¢~ I roW,
gigas and A z o m b a c t e r vinelandii. As a comparison, we also p e r f o r m e d similar m e a s u r e m e n t s on the H I P I P f r o m C h r g m a t i u m vh~osum, tn its oxidized state, this [4Fe 4S] protein also gives a weakly anisotropic E.P.R. spectrum in the g ~> 2 range, which was sometimes confused with 3Fe clusters signals.
The results o f this study e m p h a s i z e the particularly fast rela~zing properties o f the E . P R . signals in 3Fe f e r r e d e d n s .
Materials and methods ,'Pae D. gigas ferredoxin il (D.g. Fd II) sample is described in reference [2]. A. vmelandii ferredoxin I (A.v. Fd I) was purified according to the method described in [4]. The Chromatium HIPIP sample was a gift from Dr. R. Cammack, Department of Plant
They are presented on figure 2. The similarity o f the T~ values m e a s u r e d at high temperature on D.g. Fd It a n d A.v. Fd I [3] is also apparent ;n the low temperature range. They are much s l e r than those previously obtained on [2Fe-2S] [6] and [4Fe-4S] clusters [9]. It is worthwfle to c o m p a r e these properties with the relaxation b e h a v i o u r o f the [4Fe-4S] H I P I P from chromatium, which also gives a weakly anisotropic signal in the g >~ 2 ,ange in its oxidized state. The values o f T~ m e a s u r e d by continuous saturation on this s a m p l e , are in good a g r e e m e n t with the values which can be obtained from the saturation curves published in [5] (fig. 2). H o w e v e r the relaxation times T~ derived f r o m the spectral b r o a d e n i n g are significantly longer than those reporied by Blum et aL [10]. In fact these authors m e a s u r e d the b r o a d e n i n g of integrated spectra, whereas we studied the b r o a d e n i n g o f the high field part o f the derivative spectrum. Nevertheless, a careful e x a m i n a t i o n of their data reveals that the discrepancy cannot be attributed to the different methods, but is already included in the experimental results. In particular, their published spectra a p p e a r m u c h more b r o a d e n e d than those we recorded at the s a m e temperature. This c o m p a r i s o n shows that for T >~ 10 K, the
C/laraeterizat on o/' l!~ree !~'on /errede~:ins by microw~ave D .¢er saturation
k (s -! )
d~ :~'/i" •"-/' / / ". ""
109
, . , ,g ° /
10 ) !
,'
/a
At low temperature, the relaxation o f the HI PIP from C h r o m a t i u m aiso appears very efiiciem (fig. 2). As in the ,ease o f 3Fe ;'erredoxins. this ~ould resttlt from the remova! of ~he S = I / 2 degeneracy.
105
I
1
/
[
,
105 I
r
~nis explanation is compatible ,*i,'h the tempecature depender~ce o( the magnetic susceptibihty in this protelrL which can be interpreted by an Fieisenberg hamittonian ~ h e r e the difference between the J va!ues can reach l0 e r cent [1ti.
.~[/
,ol
T(K)
t ~'~(, 2
79
range of temFe<~,q~re where their E.P.R, signal can he o b s e r v e d ~his properly is certain{y related to the presence o f several low lyir~g excited states (cf. t]g. 5 o f ref. 2). |n particular, the reiaxation broadening observed for temperatures higher than 20 K i-: probably determined by the S = 3/2 states whose energy is about 50-100cm ~, whereas the ve*'y efficient telaxation below t 0 K could result trove the existence of the first S = 1 2 e~cited level ~;hose energy is only about 2*; cm : [2, 31.
2
5
10
-- [~-mpi'ramre depct~dcI!~
20
50
REFERENCES
o/ tkl ~/ava:t,,~ t m ~
1 Beinen, H g: Thorn,ion. A.J. (19t~3; 5ullur l~r,Jh'ttt~
SFe ferre&,~ins , f)~ [-d It. e 4 ~ f-d ; [4Fe-4Sl HIPIP from (hr, m*u:tlm ~m.~um . ,~k*r me:~,urcments. ,: T cadctztalcd Ire m ~h~ ~au~ ~tion cur~e:, published in vet [5}. from rot It~) ~ther irwin ,ulfur pr,:,ieiz~s .-- & {2Fc-251 {ccr~-d,~xz~ [~t,m :;p~rMina ma~tm,i [6~ -- B {~[e~tS~ fc~re,Jcxm fr:.r~ ~alll/ti~ Ml'li~¢lIil,rme)pIilh;I [91
spin-lattice relaxation in the 3Fe proteins D.g. Fd H and A.v. Fd ! is much more efficient thar~ in the other class o f iron sulfur proteins, including ~he HIP1P from C h r o m a t i u m
Discussion This study demonstrates that the 3Fe 7"erredoxins are vet 3 fast relaxing centers in the whole
Arch. ,~iochem
Biophya, 2,22, 333461. 2. Gayda, J.P., Be~rznd, P. & Th~aodu!e, F.X. (i9821 J. Chem. Ph)w., "t7. 3387-3391. 3 Ga}da. J.P., Bertrand. P.. Gaigliarelli. B. & Meyer. J. {1983~ J Chem. Phil, 79, 5732-5733. 4. Ghe,~b. D. O'DoqnelL S.. Furey. W.J.R.. Robbins. A.H. & Stout, C,D, (t982; Z MoL BioL ~58, 73. 5. Rupp. H., Rao, K.K.. D,O. & Cammack, R. (1978) B]o.him Biophy,. 4c~a. 33"/, 255-269. ~-. Gayda, J,P. Bertrand, P, De~i]le. A., More, C., Roger, G., Gibson. J-F. & Cammack. R, (]979~ [3iocbtm Biophvs. Ac~a. $1gI, 15-26. 7, Casmer, T.O. (19591 Phys, Rev.. t~5, 1506-15t5, 8. Berwan& P, Roger, G. & Gayda, J.P. t1980) ,L Magn. Res.. 4{~, 53g'-549 9. ~e~rand. P., Gasda. J P. & Rao. K.K. (i982) Z ('hem. ph, vs.. °%, 4715-4"719. i 0 g~un~, H., Sa{emo. JC., Prithee. R.C., Leig=h, 3.S. & Obnish{, 1-. U977) Bk~ph)'s .L. 20, 23-3?. il Anlanai~is. B.C & Mo~s, T.H. (1975) Bi6,chim. Biuph:,s Arm. 405, 262-279.