Cluster Characterization in Iron-Sulfur Proteins by Magnetic Circular Dichroism (Spectroscopic Probes/Ferredoxins) P

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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) respect to the number of 4-Fe clusters). Ac values are normal- spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins ized to a magnetic field of 10 kilogauss. Chromatium high-potential iron protein (HIPIP), Bacillus 1-3 MCD and stearothernophilus ferredoxin and Clostridium pasteurianum Figs. display absorption spectra for clusters ferredoxin. The MCD is found to vary significantly with cluster in the C2-, C3-, and Cl- states, respectively. The absorption oxidation state but is relatively insensitive to the nature of the spectra are typical of 4-Fe clusters, exhibiting few distinct protein. The spectra obtained are compared with the corre- features"l; for a given oxidation state the spectra are insensitive sponding spectra of iron-sulfur proteins containing 2-Fe clus- to the specific protein under study. By comparison, the MCD ters. It is concluded that MCD is useful for the characterization spectra are appreciably more structured than the absorption of iron-sulfur cluster type and oxidation state in iron-sulfur spectra but retain the insensitivity to the nature of the proteins and is superior for this purpose to absorption and nat- associated ural circular dichroism spectroscopy. protein. Most notably, the MCD spectrum of reduced HIPIP closely resembles the spectra of oxidized Bs and Cp ferredoxins, We report measurements of the magnetic circular dichroism showing that the MCD is quite insensitive to those structural (MCD) (1) of iron-sulfur proteins (2) containing 4-Fe iron- differences responsible for the very disparate redox potentials sulfur clusters, [Fe4S4(SR)4]n- (SR = protein-bound cysteine). of these proteins."* Our results (see ref. 3 for more details of these and other studies) In order of magnitude the MCD (at the field strengths used) show that: and natural CD of these proteins are comparable. However, (i) MCD is measurable throughout the near-infrared-visi- unlike the MCD and absorption spectra, the CD is highly pro- ble-ultraviolet spectral range (2000-300 nm) in all three ac- tein-dependent in form for clusters of a given oxidation state cessible oxidation states (n = 1, 2, 3; henceforth referred to as (3). C'-, C2-, and C3-). In all three oxidation states (C'-, C2-, C3-) MCD is observed (ii) Like the absorption spectra, but unlike the natural cir- down to the lowest energies (below 5000 cm'1) attained.tt cular dichroism (CD) spectra, the MCD spectrum is charac- Other features of note are the unusual monosignate nature of teristic of the cluster oxidation state and insensitive to the spe- all MCD and the comparable magnitude of the MCD in para- cific protein to which the cluster is bound. Unlike the absorption magnetic (C'-, C3-) and diamagnetic (C2-) clusters. spectra, but like the natural CD spectra, the MCD spectrum MCD has been reported for the 2-Fe iron-sulfur proteins exhibits appreciably structured features. spinach ferredoxin, adrenodoxin, and Spirulina maxima fer- (iii) The MCD spectra of the 4-Fe clusters are distinguishable redoxin (8-10). We have repeated these measurements and from those of the 2-Fe clusters [Fe2S2(SR)4]n- (n = 2, 3). extended them to include near-infrared wavelengths and to (iv) MCD is usable for the differentiation of 2-Fe and 4-Fe putidaredoxin (3). As in the 4-Fe proteins, the MCD is not very iron-sulfur clusters in iron-sulfur proteins. sensitive to the specific protein but varies with cluster oxidation MCD measurements are reported for the iron-sulfur proteins state. The 2-Fe cluster MCD spectra are distinguishably dif- Chromatium high-potential iron protein (HIPIP), Bacillus ferent from those of the 4-Fe clusters. In particular, unlike the stearothermophilus ferredoxin (Bs ferredoxin), and Clostrid- ium pasteurianum ferredoxin (Cp ferredoxin). In HIPIP the Abbreviations: MCD, magnetic circular dichroism; CD, natural circular single cluster was studied in the and dichroism; HIPIP, high-potential iron protein; Bs ferredoxin, ferredoxin C'- C2- oxidation states. from Bacillus stearothermophilus; Cp ferredoxin, ferredoxin from In Bs ferredoxin, which contains a single cluster, and in Cp Clostridium pasteurianum. ferredoxin, which contains two clusters, the C2- and C3- oxi- t Permanent address: School of Chemical Sciences, University of East dation states were studied. All measurements were made on Anglia, Norwich, England. aqueous solutions at room temperature; experiments in the * Present address: Dept. of Chemistry, University of Illinois at Chicago near-infrared required substitution of 2H20 for H20. Oxidation Circle, Chicago, IL. of reduced HIPIP was carried out by using K3Fe(CN)6. Oxi- § Present address: Dept. of Biochemistry, University of Wisconsin, dized Bs and Cp ferredoxins were Madison, WI. reduced with Na2S204. Re- 11 In addition to the well-known "390" band, all C2- state proteins ex- duced Bs and Cp ferredoxins were handled anaerobically. MCD hibit a variably resolved peak at -v9600 cm-' (-u1050 nm); this band was measured by using a Cary 61 and an infrared CD instru- has been previously reported (6) only in the 77 K spectrum of reduced ment described previously (4, 5) and at magnetic fields of ap- HIPIP. proximately 40 kilogauss (1 G = 10-4 T). Natural CD spectra ** This insensitivity of MCD to cluster environment is further dem- (3) were obtained simultaneously. The e and Ac values reported onstrated by comparison with the MCD of the synthetic analogue were calculated on a molar basis (and are not normalized with compound [Fe4S4(SCH2C6Hs)4142- shown in Fig. 1. Note the presence of the 9000 cm-1 band, previously reported (7) to be ab- sent. The publication costs of this article were defrayed in part by page tt This observation applies also to the natural CD. The CD and MCD charge payment. This article must therefore be hereby marked "ad- therefore contradict the conclusion reached from the absorption vertisement" in accordance with 18 U. S. C. §1734 solely to indicate spectrum of reduced HIPIP that low-energy transitions do not exist this fact. in the 4-Fe clusters, in contrast to 1-Fe and 2-Fe clusters. 5273 Downloaded by guest on September 28, 2021 5274 Biochemistry: Stephens et al. Proc. Natl. Acad. Sci. USA 75 (1978) A, nm A, nm 2000 1000 500 A 0.,3 / 3.0

iU .4 0.; 2.04

0.1 1.0

-- -- ...

3000

A . .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... w 2000r w 2000 20,000 w

1000 10,000

15 20 25 30 5 1 0 1 5 20 25 3 V, cm-1 X 10-3 PI cm-, X 10

FIG. 1. MCD (A) and absorption spectra (B) of: reduced Chro- FIG. 2. MCD (A) and absorption spectra (B) of: reduced Bs fer-

matium HIPIP ( ); oxidized Bs ferredoxin (.... ); oxidized Cp redoxin (- -); and reduced Cp ferredoxin (-- ferredoxin (---- ); and [N(C2H5)412[Fe4S4(SCH2C6H5)4J (in di- methylformamide).( -). Ais normalizedto a magnetic field than is possible in the more often studied visible-ultraviolet of 10 kilogauss. spectral region. For example, the 4-Fe cluster in the flavoen- zyme trimethylamine dehydrogenase, recently detected by 4-Fe proteins, no MCD is observed in the near-infrared at using the chemical extrusion technique (13), should be identi- wavelengths greater than .1000 nm. In the oxidized 2-Fe figble straightforwardly by near-infrared MCD measure- clusters, this reflects the absence of electronic transitions in this ments. region as previously demonstrated by CD measurements (11). Lastly, we note that, at the expense of the advantage of In the reduced clusters, the CD (11) shows that electronic studying solutions only at room temperature, even more de- transitions exist to well below 5000 cm-1; the lack of observation finitive characterization of iron-sulfur clusters is likely to follow of MCD here is due to the small magnitude of the MCD an- from the extension of these measurements to cryogenic (espe- isotropy ratio. cially liquid helium) temperatures (already reported for 1-Fe The MCD data obtained thus far demonstrate that MCD and 2-Fe proteins, see refs. 10 and 14). In addition to increasing combines the protein-insensitivity of absorption spectroscopy the resolution of spectral features, such measurements will allow with the more featured aspect of CD. MCD thus appears to paramagnetic and diamagnetic clusters to be more easily dis- offer a superior alternative to either of the more traditional electronic spectroscopic techniques in the characterization of A, nm 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, uti- lizing the extrusion of identifiable iron-sulfur clusters from the host protein, has recently been developed (12). At this time, 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 ad- vantages include the practicability of studying room temperature aqueous solutions of proteins in any oxidation state, diamagnetic or paramagnetic. I%# 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 FIG. 3. MCD (A) and absorption spectrum (B) of oxidized iron-sulfur proteins can lead to a more straightforward analysis Chromatium HIPIP. Downloaded by guest on September 28, 2021 Biochemistry: Stephens et al. Proc. Nat. Acad. Sci. USA 75(1978) 5275

tinguished via the temperature dependence or independence 6. Cerdonio, M., Wang, R. H., Rawlings, J. & Gray, H. B. (1974) J. of the MCD (1). 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. We gratefully acknowledge support from the National Institutes of 96,2644-2646. Health, the National Science Foundation, and the Royal Society; a gift 8. Sutherland, J., Salmeen, I., Sun, A. S. K. & Klein, M. P. (1972) of HIPIP from Professor M. D. Kamen, Dr. R. G. Bartsch, and Dr. T. Biochim. Biophys. Acta 263,550-554. E. Meyer; and a gift of [N(C2Hs)4]2[Fe4S4(SCH2C6Hs)4] from Professor 9. Ulmer, D. D., Holmquist, B. & Vallee, B. L. (1973) Biochem. R. H. Holm. 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. 1. Stephens, P. J. (1974) Annu. Rev. Phys. Chem. 25,201-232. 2. Lovenberg, W., ed. (1973, 1973, 1977) Iron-Sulfur Proteins 11. Eaton, W. A., Palmer, G., Fee, J. A., Kimura, T. & Lovenberg, (Academic, New York), Vols. 1-3. W. (1971) Proc. Natl. Acad. Sci. USA 68,3015-3020. 3. Stephens, P. J., Thomson, A. J., Dunn, J. B. R., Keiderling, T. A., 12. Holm, R. H. & Ibers, J. A. (1977) in Iron-Sulfur Proteins, ed. Rawlings, J., Rao, K. K. & Hall, D. 0. (1978) Biochemistry, in Lovenberg, W., Vol. 3, pp. 205-281. press. 13. Hill, C. L., Steenkamp, D. J., Holm, R. H. & Singer, T. P. (1977) 4. Osborne, G. A., Cheng, J. C. & Stephens, P. J. (1973) Rev. Scd. Inst. Proc. Natl. Acad. Sci. USA 74,547-551. 44, 10-15. 14. Rivoal, J. C., Briat, B., Cammack, R., Hall, D. O., Rao, K. K., 5. Nafie, L. A., Keiderling, T. A. & Stephens, P. J. (1976) J. Am. Douglas, I. N. & Thomson, A. J. (1977) Biochim. Blophys. Acta Chem. Soc. 98,2715-2722. 493, 122-131. Downloaded by guest on September 28, 2021