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The magnetic susceptibility of the oxidized and reduced iron-sulfur proteins adrenodoxin and putidaredoxin
548
SHORT COMMUNICATIONS
BBA 33232
The magnetic susceptibility of the oxidized and reduced iron-sulfur proteins adrenodoxin and putidaredoxin Nuclear hyperfine interactions1, ~ in 57Fe-labeled samples of two iron-sulfur proteins 3, adrenodoxin from beef adrenals 4, and putidaredoxin '5 from Pseudomonas putida, have shown that both irons in each of these proteins participate in a singleelectron transferring complex. We wish to report magnetic susceptibility measurements on these two proteins, which confirm chemical studies Gindicating that there is transfer of a single electron on reduction, and which suggest strong exchange coupling of the kind expected for a binuclear configuration of the two irons in the molecule. These susceptibilities follow the same pattern as those of the plant ferredoxins from spinach and parsley 7, supporting the idea that clustering of metal ions in polynuclear units may be a general phenomenon in these iron-sulfur proteins. Putidaredoxin was purified as reported previously< 8. Three independent samples were measured, 3 6 mM in iron and labile sulfur, in o.o5 M Tris HC1 buffer at pH 8.3 made o.oI M in fl-rnercaptoethanol. Adrenodoxin was purified according to published methods ~ and the sample was found to be 6. 3 mM in both iron and labile sulfur, with the purity index A41,~ rim~A28onm of o.8 7. The solvent was o.o5 M Tris (pH 8.o) made o.i M in KC1, with i ,ul of o.ox M methyl viologen added per o. 9 ml of sample to insure rapid and complete reduction 6. Samples (o. 7 ml) were measured first in the oxidized form in the above buffers and then after treatment with 25 #1 of [ M glucose and 5 #1 of approx, z mM glucose oxidase to remove dissolved oxygen. To reduce tile samples, a 2-fold molar excess of sodium dithionite was added. After completion of the susceptibility measurements on the reduced samples, iron concentration and ability to undergo reversible reduction oxidation cycles were checked by optical spectroscopic measurements on the samples. Frozen chips of the reduced adrenodoxin samples were also checked for paramagnetic impurities by X-band electron spin resonance at 77°K. None were found at a level of z % of the protein iron signal intensity. Variable temperature magnetic susceptibility measurements were made as described previously 7 with a superconducting coil vibrating sample magnetometer designed and constructed bv Dr. A. Redfield of IBM Watson Laboratory. Fig. I illustrates the susceptibility of reduced and oxidized adrenodoxin and that of a deoxygenated buffer blank. Tile diamagnetism of the oxidized form and the accurate S = I/2 Curie law paramagnetism of the reduced sample are just as were observed for the plant ferredoxins. The diamagnetism of the oxidized protein has been reported previously by KLVlr~a :~, but he reported failure to obtain reasonable values for a reduced sample. The results for tmtidaredoxin were less precise, apparently clue to the persistent presence of a small (< 2 3(~'o if high spin) am(rant of nonspecifically t)ound iron. A small paramagnetism was measured in all <~fthe oxidized samples with the exception of one which was dialyzed against o.oi M EI)TA for 24 h before the measurement (Table I). However, the EDTA treatment results in partial destruction of the iron chromaphore so that this sample cannot be verified to contain the "native" iron sulfur coordination center. Similarly, the susceptibilities of the reduced samples did l ~ i o c k i m , t ¢ i o p k y s . A c / a , _'l 4 (197 o) 54 ~ 55o
549
SHORT COMMUNICATIONS
om ~:) O.E
)O.E.j _~OA
=;-
• REDUCED PROTEIN x O X I D I Z E D PROTEIN o DEOXYGENATED BUFFER BLANK I
=ERROR BAR FOR POINTS
ALL
~
•
•
~
=
s=½'g==3"s2
o.~ 0.0
I
I
0.1
02.
I
I
-Theoreticol,i I S=O
0.3 0.4 I/T (OKI-t
0.5
0.6
0.7
Fig. I. The t e m p e r a t u r e dependence of the magnetic susceptibility of oxidized and reduced adrenodoxin.
not fit exactly to the expected values of a simple spin 1/2 system. However, the measured values were closest to S = o in the oxidized form and S = 1/2 in the reduced form so that these are taken as the most likely spin states for the 2-iron complex in the protein. The diamagnetism of the oxidized proteins again suggests that the spins of the unpaired electrons expected 3 for individual Fe(III) ions in the oxidized protein are coupled antiferromagnetically in a binuclear complex. The failure to detect any paramagnetism within the sensitivity of our apparatus up to 77°K places only a lower limit (2J >~ 6o°K) on the strength of this possible exchange coupling. In the limit of very strong coupling one must assume that at least a pair of metal electrons is in a molecular orbital encompassing both iron centers. This limiting case explanation for the susceptibility results correlates best with implications drawn from E P R experiments 1,~. The apparent hyperfine coupling constants to the two irons are nearly equal, indicating that the unpaired electron in the reduced form interacts comparably with both iron atoms. TABLE I MAGNETIC
SUSCEPTIBILITIES
OF ADRENODOXIN
AND
PUTIDAREDOXIN
dZM /*2ef f ~
(3k/N/IB 2) • [
where N is Avogadro's n u m b e r ; ZM, molar susceptibility; ,UB, Bohr m a g n e t o n ; and k, B o l t z m a n n ' s constant. For S = o the theoretical value o f / ~ 2 e f f = O. For S = 1/~ and the g values of adrenodoxin and putidaredoxin, the theoretical value of ~ e f l ' ~--- 2.86. ~t/2eff
Adrenodoxin Putidaredoxin Sample I Sample I I Sample I I After dialysis against E D T A Sample I I I
Oxidized
Reduced
o.18 ± 0.o 3
2.99 ± 0.o3
0.99 ± o.o3 0.70 ± 0.03
3.86 ± o.o 3 3.26 :k 0.03
0.22 -k 0.o5 0.45 :k 0.05
4.20 ± 0.05
Biochim. Biophys. Acta, 214 (197 o) 548-55 °
550
SHORT COMMUNICATIONS
Now that the tight coupling of iron atoms in binuclear complexes in the ironsulfur proteins is indicated by several lines of evidence, it is appropriate to ask what special biological advantages these configurations m a y confer. The most obvious way to obtain exchange coupling of the magnitude observed here and in the plant ferredoxins 7 is to require a metal-metal bond. Super-exchange, exclusively through bonds to bridging ligands, is not likely alone to provide exchange couplings as large as 5o°K (ref. IO). Theoretical studies on the molecular orbital structure of bridged systems including metal-metal bonding are thus needed. It is especially important to answer the question as to whether an isolated orbital could be generated with a range of energy sufficient to account for the wide range and extreme limits of the one electron oxidation-reduction potential observed for these proteins n. The authors would like to thank Drs. H. Beinert, L. F. Dahl, I. C. Gunsalus, and A. G. Redfield for helpful discussions as well as the use of facilities fl)r various phases of this work. This work was partially supported by Career Development Awards K3-GM-Io,236 to W.H.O.J. and K4-AM-42,386 to J.C.M.T., and Grants AM-oo562 and GM-I2394, National Institutes of Health, U.S. Public Health Service.
I B M Watson Laboratory at Columbia University, New York, N.Y. Ioo2 5 ( U . S . A . )
C. MOLESKI
T. H. Moss*
Institute for Enzyme Research, University of Wisconsin, Madison, Wisc..53706 (U.S.A.)
W . H . ORME-JOHNSON
Department of Chemistry, University of Illinois, Urbane, Ill. 618oz (U.S.A.)
J. C. M. TSIBRIS
i J. C. M. TSIBRIS, ]4,. L. TSAI, I.C. GUNSALUS, ~V. H. ORME-JOHNSON, R.F.. HANSEN AND H. BEINERT, Proc. Nell. Acad. Sci U.S., 59 (1968) 959. 2 1-I. BEINERT AND X,¥. t{. ORME-JOHNSON, Ann. N . Y . Acad. Sci., 158 (1969) 366. 3 J- C. M. TSlBRIS AND R. W. W'OODY, Coord. Chem. Rev., in the press. 4 T. ()MURA, E. ~ANDERS, R . ~V. ESTABROOK, D. Y . COOPER AND O. ROSENTHAL, Arch. Biochem. Biophys., t l 7 (I966) 66o. 5 ])" W . CUSHMAN, R. L, TSAI AND l, C. (~UNSALUS, Biochem. Biophys. Res. Commun., 2(5 (1967) 5 7 7 . 6 \V. H. ORME-JOHNSON AND H. BEINERT, J . Biol. Chem., 244 (1969) 6143; and references therein. 7 T. H. Moss, D. PFTERING AND (~-. PALMER, J. ~iol. Chem., 244 (1969) 2275. 8 R . C. TSAI,
j.C.M.
1{. [;. NANSEN
ANI)
H. BFINERT, in preparation. 9 T. KIMURA, Abstr. 3rd Intern. :lJeeting on Magnetic Resonance in Biology, ll'arrcnton,
TSIBRIS,
I . C . (;UNSALUS,
W . H . ORME-JOHNSON,
l'a.,
r967.
io J. B. GOODENOUGH, Magnetism and the Chemical Bond, lnterscience, New York, 1963, p. 18o. i i 1). (). HALL AND M. C. W. ]'2VANS, Nature, 223 (1969) 134:. I2 R. MALKIN AND J. C. RABINOWlTZ, Ann. Rev. Biochem., 36 (1967) 113.
Received April 2oth, I97O * To w h o m reprint requests should be addressed.
Biochim. Biophys. Acta, 214 (I97 o) 548-55 o
SHORT COMMUNICATIONS
BBA 33232
The magnetic susceptibility of the oxidized and reduced iron-sulfur proteins adrenodoxin and putidaredoxin Nuclear hyperfine interactions1, ~ in 57Fe-labeled samples of two iron-sulfur proteins 3, adrenodoxin from beef adrenals 4, and putidaredoxin '5 from Pseudomonas putida, have shown that both irons in each of these proteins participate in a singleelectron transferring complex. We wish to report magnetic susceptibility measurements on these two proteins, which confirm chemical studies Gindicating that there is transfer of a single electron on reduction, and which suggest strong exchange coupling of the kind expected for a binuclear configuration of the two irons in the molecule. These susceptibilities follow the same pattern as those of the plant ferredoxins from spinach and parsley 7, supporting the idea that clustering of metal ions in polynuclear units may be a general phenomenon in these iron-sulfur proteins. Putidaredoxin was purified as reported previously< 8. Three independent samples were measured, 3 6 mM in iron and labile sulfur, in o.o5 M Tris HC1 buffer at pH 8.3 made o.oI M in fl-rnercaptoethanol. Adrenodoxin was purified according to published methods ~ and the sample was found to be 6. 3 mM in both iron and labile sulfur, with the purity index A41,~ rim~A28onm of o.8 7. The solvent was o.o5 M Tris (pH 8.o) made o.i M in KC1, with i ,ul of o.ox M methyl viologen added per o. 9 ml of sample to insure rapid and complete reduction 6. Samples (o. 7 ml) were measured first in the oxidized form in the above buffers and then after treatment with 25 #1 of [ M glucose and 5 #1 of approx, z mM glucose oxidase to remove dissolved oxygen. To reduce tile samples, a 2-fold molar excess of sodium dithionite was added. After completion of the susceptibility measurements on the reduced samples, iron concentration and ability to undergo reversible reduction oxidation cycles were checked by optical spectroscopic measurements on the samples. Frozen chips of the reduced adrenodoxin samples were also checked for paramagnetic impurities by X-band electron spin resonance at 77°K. None were found at a level of z % of the protein iron signal intensity. Variable temperature magnetic susceptibility measurements were made as described previously 7 with a superconducting coil vibrating sample magnetometer designed and constructed bv Dr. A. Redfield of IBM Watson Laboratory. Fig. I illustrates the susceptibility of reduced and oxidized adrenodoxin and that of a deoxygenated buffer blank. Tile diamagnetism of the oxidized form and the accurate S = I/2 Curie law paramagnetism of the reduced sample are just as were observed for the plant ferredoxins. The diamagnetism of the oxidized protein has been reported previously by KLVlr~a :~, but he reported failure to obtain reasonable values for a reduced sample. The results for tmtidaredoxin were less precise, apparently clue to the persistent presence of a small (< 2 3(~'o if high spin) am(rant of nonspecifically t)ound iron. A small paramagnetism was measured in all <~fthe oxidized samples with the exception of one which was dialyzed against o.oi M EI)TA for 24 h before the measurement (Table I). However, the EDTA treatment results in partial destruction of the iron chromaphore so that this sample cannot be verified to contain the "native" iron sulfur coordination center. Similarly, the susceptibilities of the reduced samples did l ~ i o c k i m , t ¢ i o p k y s . A c / a , _'l 4 (197 o) 54 ~ 55o
549
SHORT COMMUNICATIONS
om ~:) O.E
)O.E.j _~OA
=;-
• REDUCED PROTEIN x O X I D I Z E D PROTEIN o DEOXYGENATED BUFFER BLANK I
=ERROR BAR FOR POINTS
ALL
~
•
•
~
=
s=½'g==3"s2
o.~ 0.0
I
I
0.1
02.
I
I
-Theoreticol,i I S=O
0.3 0.4 I/T (OKI-t
0.5
0.6
0.7
Fig. I. The t e m p e r a t u r e dependence of the magnetic susceptibility of oxidized and reduced adrenodoxin.
not fit exactly to the expected values of a simple spin 1/2 system. However, the measured values were closest to S = o in the oxidized form and S = 1/2 in the reduced form so that these are taken as the most likely spin states for the 2-iron complex in the protein. The diamagnetism of the oxidized proteins again suggests that the spins of the unpaired electrons expected 3 for individual Fe(III) ions in the oxidized protein are coupled antiferromagnetically in a binuclear complex. The failure to detect any paramagnetism within the sensitivity of our apparatus up to 77°K places only a lower limit (2J >~ 6o°K) on the strength of this possible exchange coupling. In the limit of very strong coupling one must assume that at least a pair of metal electrons is in a molecular orbital encompassing both iron centers. This limiting case explanation for the susceptibility results correlates best with implications drawn from E P R experiments 1,~. The apparent hyperfine coupling constants to the two irons are nearly equal, indicating that the unpaired electron in the reduced form interacts comparably with both iron atoms. TABLE I MAGNETIC
SUSCEPTIBILITIES
OF ADRENODOXIN
AND
PUTIDAREDOXIN
dZM /*2ef f ~
(3k/N/IB 2) • [
where N is Avogadro's n u m b e r ; ZM, molar susceptibility; ,UB, Bohr m a g n e t o n ; and k, B o l t z m a n n ' s constant. For S = o the theoretical value o f / ~ 2 e f f = O. For S = 1/~ and the g values of adrenodoxin and putidaredoxin, the theoretical value of ~ e f l ' ~--- 2.86. ~t/2eff
Adrenodoxin Putidaredoxin Sample I Sample I I Sample I I After dialysis against E D T A Sample I I I
Oxidized
Reduced
o.18 ± 0.o 3
2.99 ± 0.o3
0.99 ± o.o3 0.70 ± 0.03
3.86 ± o.o 3 3.26 :k 0.03
0.22 -k 0.o5 0.45 :k 0.05
4.20 ± 0.05
Biochim. Biophys. Acta, 214 (197 o) 548-55 °
550
SHORT COMMUNICATIONS
Now that the tight coupling of iron atoms in binuclear complexes in the ironsulfur proteins is indicated by several lines of evidence, it is appropriate to ask what special biological advantages these configurations m a y confer. The most obvious way to obtain exchange coupling of the magnitude observed here and in the plant ferredoxins 7 is to require a metal-metal bond. Super-exchange, exclusively through bonds to bridging ligands, is not likely alone to provide exchange couplings as large as 5o°K (ref. IO). Theoretical studies on the molecular orbital structure of bridged systems including metal-metal bonding are thus needed. It is especially important to answer the question as to whether an isolated orbital could be generated with a range of energy sufficient to account for the wide range and extreme limits of the one electron oxidation-reduction potential observed for these proteins n. The authors would like to thank Drs. H. Beinert, L. F. Dahl, I. C. Gunsalus, and A. G. Redfield for helpful discussions as well as the use of facilities fl)r various phases of this work. This work was partially supported by Career Development Awards K3-GM-Io,236 to W.H.O.J. and K4-AM-42,386 to J.C.M.T., and Grants AM-oo562 and GM-I2394, National Institutes of Health, U.S. Public Health Service.
I B M Watson Laboratory at Columbia University, New York, N.Y. Ioo2 5 ( U . S . A . )
C. MOLESKI
T. H. Moss*
Institute for Enzyme Research, University of Wisconsin, Madison, Wisc..53706 (U.S.A.)
W . H . ORME-JOHNSON
Department of Chemistry, University of Illinois, Urbane, Ill. 618oz (U.S.A.)
J. C. M. TSIBRIS
i J. C. M. TSIBRIS, ]4,. L. TSAI, I.C. GUNSALUS, ~V. H. ORME-JOHNSON, R.F.. HANSEN AND H. BEINERT, Proc. Nell. Acad. Sci U.S., 59 (1968) 959. 2 1-I. BEINERT AND X,¥. t{. ORME-JOHNSON, Ann. N . Y . Acad. Sci., 158 (1969) 366. 3 J- C. M. TSlBRIS AND R. W. W'OODY, Coord. Chem. Rev., in the press. 4 T. ()MURA, E. ~ANDERS, R . ~V. ESTABROOK, D. Y . COOPER AND O. ROSENTHAL, Arch. Biochem. Biophys., t l 7 (I966) 66o. 5 ])" W . CUSHMAN, R. L, TSAI AND l, C. (~UNSALUS, Biochem. Biophys. Res. Commun., 2(5 (1967) 5 7 7 . 6 \V. H. ORME-JOHNSON AND H. BEINERT, J . Biol. Chem., 244 (1969) 6143; and references therein. 7 T. H. Moss, D. PFTERING AND (~-. PALMER, J. ~iol. Chem., 244 (1969) 2275. 8 R . C. TSAI,
j.C.M.
1{. [;. NANSEN
ANI)
H. BFINERT, in preparation. 9 T. KIMURA, Abstr. 3rd Intern. :lJeeting on Magnetic Resonance in Biology, ll'arrcnton,
TSIBRIS,
I . C . (;UNSALUS,
W . H . ORME-JOHNSON,
l'a.,
r967.
io J. B. GOODENOUGH, Magnetism and the Chemical Bond, lnterscience, New York, 1963, p. 18o. i i 1). (). HALL AND M. C. W. ]'2VANS, Nature, 223 (1969) 134:. I2 R. MALKIN AND J. C. RABINOWlTZ, Ann. Rev. Biochem., 36 (1967) 113.
Received April 2oth, I97O * To w h o m reprint requests should be addressed.
Biochim. Biophys. Acta, 214 (I97 o) 548-55 o