Proc. Natl. Acad. Sci. USA Vol. 75, No. 11, pp. 5273-5275, November 1978
Biochemistry
Cluster characterization in iron-sulfur proteins by magnetic circular dichroism (spectroscopic probes/ferredoxins)
P. J. STEPHENS*, A. J. THOMSON*t, T. A.
KEIDERLING*t, J. RAWLINGS*§, K. K. RAOT, AND D. 0. HALLS
Department of Chemistry, University of Southern California, Los Angeles, California 90007; and ISchool of Biological Sciences, University of London King's College, 68 Half Moon Lane, London, England *
Communicated by Martin D. Kamen, August 2,1978-
ABSTRACT We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothernophilus ferredoxin and Clostridium pasteurianum ferredoxin. The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein. The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters. It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and natural circular dichroism spectroscopy.
We report measurements of the magnetic circular dichroism (MCD) (1) of iron-sulfur proteins (2) containing 4-Fe ironsulfur clusters, [Fe4S4(SR)4]n- (SR = protein-bound cysteine). Our results (see ref. 3 for more details of these and other studies) show that: (i) MCD is measurable throughout the near-infrared-visible-ultraviolet spectral range (2000-300 nm) in all three accessible oxidation states (n = 1, 2, 3; henceforth referred to as C'-, C2-, and C3-). (ii) Like the absorption spectra, but unlike the natural circular dichroism (CD) spectra, the MCD spectrum is characteristic of the cluster oxidation state and insensitive to the specific protein to which the cluster is bound. Unlike the absorption spectra, but like the natural CD spectra, the MCD spectrum exhibits appreciably structured features. (iii) The MCD spectra of the 4-Fe clusters are distinguishable from those of the 2-Fe clusters [Fe2S2(SR)4]n- (n = 2, 3). (iv) MCD is usable for the differentiation of 2-Fe and 4-Fe iron-sulfur clusters in iron-sulfur proteins. MCD measurements are reported for the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin (Bs ferredoxin), and Clostridium pasteurianum ferredoxin (Cp ferredoxin). In HIPIP the single cluster was studied in the C'- and C2- oxidation states. In Bs ferredoxin, which contains a single cluster, and in Cp ferredoxin, which contains two clusters, the C2- and C3- oxidation states were studied. All measurements were made on aqueous solutions at room temperature; experiments in the near-infrared required substitution of 2H20 for H20. Oxidation of reduced HIPIP was carried out by using K3Fe(CN)6. Oxidized Bs and Cp ferredoxins were reduced with Na2S204. Reduced Bs and Cp ferredoxins were handled anaerobically. MCD was measured by using a Cary 61 and an infrared CD instrument described previously (4, 5) and at magnetic fields of approximately 40 kilogauss (1 G = 10-4 T). Natural CD spectra (3) were obtained simultaneously. The e and Ac values reported were calculated on a molar basis (and are not normalized with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5273
respect to the number of 4-Fe clusters). Ac values are normalized to a magnetic field of 10 kilogauss. Figs. 1-3 display MCD and absorption spectra for clusters in the C2-, C3-, and Cl- states, respectively. The absorption spectra are typical of 4-Fe clusters, exhibiting few distinct features"l; for a given oxidation state the spectra are insensitive to the specific protein under study. By comparison, the MCD spectra are appreciably more structured than the absorption spectra but retain the insensitivity to the nature of the associated protein. Most notably, the MCD spectrum of reduced HIPIP closely resembles the spectra of oxidized Bs and Cp ferredoxins, showing that the MCD is quite insensitive to those structural differences responsible for the very disparate redox potentials of these proteins."* In order of magnitude the MCD (at the field strengths used) and natural CD of these proteins are comparable. However, unlike the MCD and absorption spectra, the CD is highly protein-dependent in form for clusters of a given oxidation state (3). In all three oxidation states (C'-, C2-, C3-) MCD is observed down to the lowest energies (below 5000 cm'1) attained.tt Other features of note are the unusual monosignate nature of all MCD and the comparable magnitude of the MCD in paramagnetic (C'-, C3-) and diamagnetic (C2-) clusters. MCD has been reported for the 2-Fe iron-sulfur proteins spinach ferredoxin, adrenodoxin, and Spirulina maxima ferredoxin (8-10). We have repeated these measurements and extended them to include near-infrared wavelengths and to putidaredoxin (3). As in the 4-Fe proteins, the MCD is not very sensitive to the specific protein but varies with cluster oxidation state. The 2-Fe cluster MCD spectra are distinguishably different from those of the 4-Fe clusters. In particular, unlike the Abbreviations: MCD, magnetic circular dichroism; CD, natural circular dichroism; HIPIP, high-potential iron protein; Bs ferredoxin, ferredoxin from Bacillus stearothermophilus; Cp ferredoxin, ferredoxin from Clostridium pasteurianum. t Permanent address: School of Chemical Sciences, University of East Anglia, Norwich, England. * Present address: Dept. of Chemistry, University of Illinois at Chicago Circle, Chicago, IL. § Present address: Dept. of Biochemistry, University of Wisconsin,
Madison, WI. 1 In addition to the well-known "390" band, all C2- state proteins exhibit a variably resolved peak at -v9600 cm-' (-u1050 nm); this band has been previously reported (6) only in the 77 K spectrum of reduced HIPIP. ** This insensitivity of MCD to cluster environment is further demonstrated by comparison with the MCD of the synthetic analogue compound [Fe4S4(SCH2C6Hs)4142- shown in Fig. 1. Note the presence of the 9000 cm-1 band, previously reported (7) to be absent. tt This observation applies also to the natural CD. The CD and MCD therefore contradict the conclusion reached from the absorption spectrum of reduced HIPIP that low-energy transitions do not exist in the 4-Fe clusters, in contrast to 1-Fe and 2-Fe clusters.
5274
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Stephens et al.
A, nm
A, nm 2000
500
1000
A
/
0.,3 iU .4
3.0
0.;
2.04 1.0
0.1 --
--
...
3000 w 2000r
.~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .
A
w 2000
...
20,000 10,000
1000
20 25 15 cm-1 X 10-3
30
5
10
FIG. 1. MCD (A) and absorption spectra (B) of: reduced Chromatium HIPIP ( ); oxidized Bs ferredoxin ( ); oxidized Cp ferredoxin (---- ); and [N(C2H5)412[Fe4S4(SCH2C6H5)4J (in di-). Ais normalizedto a magnetic field methylformamide).( of 10 kilogauss. ....
4-Fe proteins, no MCD is observed in the near-infrared at wavelengths greater than .1000 nm. In the oxidized 2-Fe clusters, this reflects the absence of electronic transitions in this region as previously demonstrated by CD measurements (11). In the reduced clusters, the CD (11) shows that electronic transitions exist to well below 5000 cm-1; the lack of observation of MCD here is due to the small magnitude of the MCD anisotropy ratio. The MCD data obtained thus far demonstrate that MCD combines the protein-insensitivity of absorption spectroscopy with the more featured aspect of CD. MCD thus appears to offer a superior alternative to either of the more traditional electronic spectroscopic techniques in the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins. Many other spectroscopic techniques have been applied to the study of iron-sulfur proteins (2), particularly electron paramagnetic resonance, Mbssbauer, proton NMR, and resonance Raman techniques. In addition, a powerful chemical technique for the characterization of iron-sulfur clusters, utilizing the extrusion of identifiable iron-sulfur clusters from the host protein, has recently been developed (12). At this time,
20
15 PI
V,
w
25
3
cm-, X 10
FIG. 2. MCD (A) and absorption spectra (B) of: reduced Bs ferredoxin (- -); and reduced Cp ferredoxin (--
than is possible in the more often studied visible-ultraviolet spectral region. For example, the 4-Fe cluster in the flavoenzyme trimethylamine dehydrogenase, recently detected by using the chemical extrusion technique (13), should be identifigble straightforwardly by near-infrared MCD measurements.
Lastly, we note that, at the expense of the advantage of studying solutions only at room temperature, even more definitive characterization of iron-sulfur clusters is likely to follow from the extension of these measurements to cryogenic (especially liquid helium) temperatures (already reported for 1-Fe and 2-Fe proteins, see refs. 10 and 14). In addition to increasing the resolution of spectral features, such measurements will allow paramagnetic and diamagnetic clusters to be more easily disA,
nm
however, no one technique can be used routinely to identify unambiguously all cluster types and oxidation states in a complex iron-sulfur protein. MCD therefore appears to be of some potential utility for cluster characterization. Its particular advantages include the practicability of studying room temperature aqueous solutions of proteins in any oxidation state, diamagnetic or paramagnetic. In the application of optical spectroscopy to the study of iron-sulfur clusters in complex proteins, additional difficulties are introduced when other chromophoric entities (prosthetic groups)-such as flavins, hemes, or other transition metal ions-are present. Many such interfering groups are devoid of electronic transitions in the near-infrared, however, and in such systems the existence of spectra in the near-infrared region in iron-sulfur proteins can lead to a more straightforward analysis
I%#
FIG. 3. MCD (A) and absorption spectrum (B) of oxidized Chromatium HIPIP.
Biochemistry: Stephens et al. tinguished via the temperature dependence or independence of the MCD (1). We gratefully acknowledge support from the National Institutes of Health, the National Science Foundation, and the Royal Society; a gift of HIPIP from Professor M. D. Kamen, Dr. R. G. Bartsch, and Dr. T. E. Meyer; and a gift of [N(C2Hs)4]2[Fe4S4(SCH2C6Hs)4] from Professor R. H. Holm. 1. Stephens, P. J. (1974) Annu. Rev. Phys. Chem. 25,201-232. 2. Lovenberg, W., ed. (1973, 1973, 1977) Iron-Sulfur Proteins (Academic, New York), Vols. 1-3. 3. Stephens, P. J., Thomson, A. J., Dunn, J. B. R., Keiderling, T. A., Rawlings, J., Rao, K. K. & Hall, D. 0. (1978) Biochemistry, in
press.
4. Osborne, G. A., Cheng, J. C. & Stephens, P. J. (1973) Rev. Scd. Inst. 44, 10-15. 5. Nafie, L. A., Keiderling, T. A. & Stephens, P. J. (1976) J. Am. Chem. Soc. 98,2715-2722.
Proc. Nat. Acad. Sci. USA 75(1978)
5275
6. Cerdonio, M., Wang, R. H., Rawlings, J. & Gray, H. B. (1974) J. Am. Chem. Soc. 96,6534-6535. 7. Holm, R. H., Averill, B. A., Herskovitz, T., Frankel, R. B., Gray, H. B., Siiman, 0. & Grunthaner, F. J. (1974) J. Am. Chem. Soc. 96,2644-2646. 8. Sutherland, J., Salmeen, I., Sun, A. S. K. & Klein, M. P. (1972) Biochim. Biophys. Acta 263,550-554. 9. Ulmer, D. D., Holmquist, B. & Vallee, B. L. (1973) Biochem. Biophys. Res. Commun. 51, 1054-1061. 10. Thomson, A. J., Cammack, R., Hall, D. O., Rao, K. K., Briat, B., Rivoal, J. C. & Badoz, J. (1977) Biochim. Biophys. Acta 493, 132-141. 11. Eaton, W. A., Palmer, G., Fee, J. A., Kimura, T. & Lovenberg,
W. (1971) Proc. Natl. Acad. Sci. USA 68,3015-3020. 12. Holm, R. H. & Ibers, J. A. (1977) in Iron-Sulfur Proteins, ed. Lovenberg, W., Vol. 3, pp. 205-281. 13. Hill, C. L., Steenkamp, D. J., Holm, R. H. & Singer, T. P. (1977)
Proc. Natl. Acad. Sci. USA 74,547-551. 14. Rivoal, J. C., Briat, B., Cammack, R., Hall, D. O., Rao, K. K., Douglas, I. N. & Thomson, A. J. (1977) Biochim. Blophys. Acta
493, 122-131.
Biochemistry
Cluster characterization in iron-sulfur proteins by magnetic circular dichroism (spectroscopic probes/ferredoxins)
P. J. STEPHENS*, A. J. THOMSON*t, T. A.
KEIDERLING*t, J. RAWLINGS*§, K. K. RAOT, AND D. 0. HALLS
Department of Chemistry, University of Southern California, Los Angeles, California 90007; and ISchool of Biological Sciences, University of London King's College, 68 Half Moon Lane, London, England *
Communicated by Martin D. Kamen, August 2,1978-
ABSTRACT We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothernophilus ferredoxin and Clostridium pasteurianum ferredoxin. The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein. The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters. It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and natural circular dichroism spectroscopy.
We report measurements of the magnetic circular dichroism (MCD) (1) of iron-sulfur proteins (2) containing 4-Fe ironsulfur clusters, [Fe4S4(SR)4]n- (SR = protein-bound cysteine). Our results (see ref. 3 for more details of these and other studies) show that: (i) MCD is measurable throughout the near-infrared-visible-ultraviolet spectral range (2000-300 nm) in all three accessible oxidation states (n = 1, 2, 3; henceforth referred to as C'-, C2-, and C3-). (ii) Like the absorption spectra, but unlike the natural circular dichroism (CD) spectra, the MCD spectrum is characteristic of the cluster oxidation state and insensitive to the specific protein to which the cluster is bound. Unlike the absorption spectra, but like the natural CD spectra, the MCD spectrum exhibits appreciably structured features. (iii) The MCD spectra of the 4-Fe clusters are distinguishable from those of the 2-Fe clusters [Fe2S2(SR)4]n- (n = 2, 3). (iv) MCD is usable for the differentiation of 2-Fe and 4-Fe iron-sulfur clusters in iron-sulfur proteins. MCD measurements are reported for the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin (Bs ferredoxin), and Clostridium pasteurianum ferredoxin (Cp ferredoxin). In HIPIP the single cluster was studied in the C'- and C2- oxidation states. In Bs ferredoxin, which contains a single cluster, and in Cp ferredoxin, which contains two clusters, the C2- and C3- oxidation states were studied. All measurements were made on aqueous solutions at room temperature; experiments in the near-infrared required substitution of 2H20 for H20. Oxidation of reduced HIPIP was carried out by using K3Fe(CN)6. Oxidized Bs and Cp ferredoxins were reduced with Na2S204. Reduced Bs and Cp ferredoxins were handled anaerobically. MCD was measured by using a Cary 61 and an infrared CD instrument described previously (4, 5) and at magnetic fields of approximately 40 kilogauss (1 G = 10-4 T). Natural CD spectra (3) were obtained simultaneously. The e and Ac values reported were calculated on a molar basis (and are not normalized with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5273
respect to the number of 4-Fe clusters). Ac values are normalized to a magnetic field of 10 kilogauss. Figs. 1-3 display MCD and absorption spectra for clusters in the C2-, C3-, and Cl- states, respectively. The absorption spectra are typical of 4-Fe clusters, exhibiting few distinct features"l; for a given oxidation state the spectra are insensitive to the specific protein under study. By comparison, the MCD spectra are appreciably more structured than the absorption spectra but retain the insensitivity to the nature of the associated protein. Most notably, the MCD spectrum of reduced HIPIP closely resembles the spectra of oxidized Bs and Cp ferredoxins, showing that the MCD is quite insensitive to those structural differences responsible for the very disparate redox potentials of these proteins."* In order of magnitude the MCD (at the field strengths used) and natural CD of these proteins are comparable. However, unlike the MCD and absorption spectra, the CD is highly protein-dependent in form for clusters of a given oxidation state (3). In all three oxidation states (C'-, C2-, C3-) MCD is observed down to the lowest energies (below 5000 cm'1) attained.tt Other features of note are the unusual monosignate nature of all MCD and the comparable magnitude of the MCD in paramagnetic (C'-, C3-) and diamagnetic (C2-) clusters. MCD has been reported for the 2-Fe iron-sulfur proteins spinach ferredoxin, adrenodoxin, and Spirulina maxima ferredoxin (8-10). We have repeated these measurements and extended them to include near-infrared wavelengths and to putidaredoxin (3). As in the 4-Fe proteins, the MCD is not very sensitive to the specific protein but varies with cluster oxidation state. The 2-Fe cluster MCD spectra are distinguishably different from those of the 4-Fe clusters. In particular, unlike the Abbreviations: MCD, magnetic circular dichroism; CD, natural circular dichroism; HIPIP, high-potential iron protein; Bs ferredoxin, ferredoxin from Bacillus stearothermophilus; Cp ferredoxin, ferredoxin from Clostridium pasteurianum. t Permanent address: School of Chemical Sciences, University of East Anglia, Norwich, England. * Present address: Dept. of Chemistry, University of Illinois at Chicago Circle, Chicago, IL. § Present address: Dept. of Biochemistry, University of Wisconsin,
Madison, WI. 1 In addition to the well-known "390" band, all C2- state proteins exhibit a variably resolved peak at -v9600 cm-' (-u1050 nm); this band has been previously reported (6) only in the 77 K spectrum of reduced HIPIP. ** This insensitivity of MCD to cluster environment is further demonstrated by comparison with the MCD of the synthetic analogue compound [Fe4S4(SCH2C6Hs)4142- shown in Fig. 1. Note the presence of the 9000 cm-1 band, previously reported (7) to be absent. tt This observation applies also to the natural CD. The CD and MCD therefore contradict the conclusion reached from the absorption spectrum of reduced HIPIP that low-energy transitions do not exist in the 4-Fe clusters, in contrast to 1-Fe and 2-Fe clusters.
5274
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Stephens et al.
A, nm
A, nm 2000
500
1000
A
/
0.,3 iU .4
3.0
0.;
2.04 1.0
0.1 --
--
...
3000 w 2000r
.~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .
A
w 2000
...
20,000 10,000
1000
20 25 15 cm-1 X 10-3
30
5
10
FIG. 1. MCD (A) and absorption spectra (B) of: reduced Chromatium HIPIP ( ); oxidized Bs ferredoxin ( ); oxidized Cp ferredoxin (---- ); and [N(C2H5)412[Fe4S4(SCH2C6H5)4J (in di-). Ais normalizedto a magnetic field methylformamide).( of 10 kilogauss. ....
4-Fe proteins, no MCD is observed in the near-infrared at wavelengths greater than .1000 nm. In the oxidized 2-Fe clusters, this reflects the absence of electronic transitions in this region as previously demonstrated by CD measurements (11). In the reduced clusters, the CD (11) shows that electronic transitions exist to well below 5000 cm-1; the lack of observation of MCD here is due to the small magnitude of the MCD anisotropy ratio. The MCD data obtained thus far demonstrate that MCD combines the protein-insensitivity of absorption spectroscopy with the more featured aspect of CD. MCD thus appears to offer a superior alternative to either of the more traditional electronic spectroscopic techniques in the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins. Many other spectroscopic techniques have been applied to the study of iron-sulfur proteins (2), particularly electron paramagnetic resonance, Mbssbauer, proton NMR, and resonance Raman techniques. In addition, a powerful chemical technique for the characterization of iron-sulfur clusters, utilizing the extrusion of identifiable iron-sulfur clusters from the host protein, has recently been developed (12). At this time,
20
15 PI
V,
w
25
3
cm-, X 10
FIG. 2. MCD (A) and absorption spectra (B) of: reduced Bs ferredoxin (- -); and reduced Cp ferredoxin (--
than is possible in the more often studied visible-ultraviolet spectral region. For example, the 4-Fe cluster in the flavoenzyme trimethylamine dehydrogenase, recently detected by using the chemical extrusion technique (13), should be identifigble straightforwardly by near-infrared MCD measurements.
Lastly, we note that, at the expense of the advantage of studying solutions only at room temperature, even more definitive characterization of iron-sulfur clusters is likely to follow from the extension of these measurements to cryogenic (especially liquid helium) temperatures (already reported for 1-Fe and 2-Fe proteins, see refs. 10 and 14). In addition to increasing the resolution of spectral features, such measurements will allow paramagnetic and diamagnetic clusters to be more easily disA,
nm
however, no one technique can be used routinely to identify unambiguously all cluster types and oxidation states in a complex iron-sulfur protein. MCD therefore appears to be of some potential utility for cluster characterization. Its particular advantages include the practicability of studying room temperature aqueous solutions of proteins in any oxidation state, diamagnetic or paramagnetic. In the application of optical spectroscopy to the study of iron-sulfur clusters in complex proteins, additional difficulties are introduced when other chromophoric entities (prosthetic groups)-such as flavins, hemes, or other transition metal ions-are present. Many such interfering groups are devoid of electronic transitions in the near-infrared, however, and in such systems the existence of spectra in the near-infrared region in iron-sulfur proteins can lead to a more straightforward analysis
I%#
FIG. 3. MCD (A) and absorption spectrum (B) of oxidized Chromatium HIPIP.
Biochemistry: Stephens et al. tinguished via the temperature dependence or independence of the MCD (1). We gratefully acknowledge support from the National Institutes of Health, the National Science Foundation, and the Royal Society; a gift of HIPIP from Professor M. D. Kamen, Dr. R. G. Bartsch, and Dr. T. E. Meyer; and a gift of [N(C2Hs)4]2[Fe4S4(SCH2C6Hs)4] from Professor R. H. Holm. 1. Stephens, P. J. (1974) Annu. Rev. Phys. Chem. 25,201-232. 2. Lovenberg, W., ed. (1973, 1973, 1977) Iron-Sulfur Proteins (Academic, New York), Vols. 1-3. 3. Stephens, P. J., Thomson, A. J., Dunn, J. B. R., Keiderling, T. A., Rawlings, J., Rao, K. K. & Hall, D. 0. (1978) Biochemistry, in
press.
4. Osborne, G. A., Cheng, J. C. & Stephens, P. J. (1973) Rev. Scd. Inst. 44, 10-15. 5. Nafie, L. A., Keiderling, T. A. & Stephens, P. J. (1976) J. Am. Chem. Soc. 98,2715-2722.
Proc. Nat. Acad. Sci. USA 75(1978)
5275
6. Cerdonio, M., Wang, R. H., Rawlings, J. & Gray, H. B. (1974) J. Am. Chem. Soc. 96,6534-6535. 7. Holm, R. H., Averill, B. A., Herskovitz, T., Frankel, R. B., Gray, H. B., Siiman, 0. & Grunthaner, F. J. (1974) J. Am. Chem. Soc. 96,2644-2646. 8. Sutherland, J., Salmeen, I., Sun, A. S. K. & Klein, M. P. (1972) Biochim. Biophys. Acta 263,550-554. 9. Ulmer, D. D., Holmquist, B. & Vallee, B. L. (1973) Biochem. Biophys. Res. Commun. 51, 1054-1061. 10. Thomson, A. J., Cammack, R., Hall, D. O., Rao, K. K., Briat, B., Rivoal, J. C. & Badoz, J. (1977) Biochim. Biophys. Acta 493, 132-141. 11. Eaton, W. A., Palmer, G., Fee, J. A., Kimura, T. & Lovenberg,
W. (1971) Proc. Natl. Acad. Sci. USA 68,3015-3020. 12. Holm, R. H. & Ibers, J. A. (1977) in Iron-Sulfur Proteins, ed. Lovenberg, W., Vol. 3, pp. 205-281. 13. Hill, C. L., Steenkamp, D. J., Holm, R. H. & Singer, T. P. (1977)
Proc. Natl. Acad. Sci. USA 74,547-551. 14. Rivoal, J. C., Briat, B., Cammack, R., Hall, D. O., Rao, K. K., Douglas, I. N. & Thomson, A. J. (1977) Biochim. Blophys. Acta
493, 122-131.
