A Gallium-Substituted Cubane-Type Cluster in Pyrococcus furiosus Ferredoxin Keith A. Johnson, ‡ Phillip S. Brereton, ² Marc F. J. M. Verhagen, ² Luigi Calzolai, § Gerd N. La Mar, § Michael W. W. Adams,* ,² and I. Jonathan Amster ‡ Department of Chemistry and Department of Biochemistry and Molecular Biology UniVersity of Georgia, Athens, Georgia 30602 Department of Chemistry, UniVersity of California, DaVis DaVis, California 95616 ReceiVed April 25, 2001 Iron-sulfur (Fe-S) clusters are ubiquitous in nature and play a variety of roles including electron transfer and catalysis. 1 One of the best studied is the ferredoxin (Fd) from Pyrococcus furiosus (Pf). Pf Fd contains a single [4Fe-4S] cluster that is coordinated to the polypeptide chain by three cysteinyl and one aspartyl ligand. The high stability of this protein made it an extremely useful model system to obtain various cluster types. For example, the native 4Fe cluster is readily converted in vitro to a [3Fe-4S] form by oxidative loss of the Fe atom ligated by the aspartyl ligand. 2 Interestingly, there are no significant differences between the three-dimensional structures of Pf Fd with a [3Fe-4S] cluster or a [4Fe-4S] cluster. 3,4 Moreover, addition of various types of metal ion (M) to the 3Fe-form yields various mixed-metal clusters [M3Fe-4S] within this protein. 5 However, there have been very few reports on replacing all the Fe atoms in a metal-containing center. Substitution of Cd(II), Co(II), and Ru(II) into [2Fe-2S] and 2[4Fe-4S] Fds was reported but the characterization of these products did not conclusively determine the nature of the resulting metal centers. 6 Isomorphous replacement of ferric for gallium- (III) atoms in a [2Fe-2S] putidaredoxin converted it into a protein containing a [Ga-4S] center, effectively a rubredoxin-like metal center. 7 The first example of a [2Ga-2S] Fd was prepared from Anabaena 7120 Fd. 8 The tertiary structures of the native proteins in the latter two instances were maintained. Here we provide evidence for the first characterized all-Ga cubane-type cluster in an Fe-S protein. The wild-type and D14C mutant forms of Pf Fd were used in this study. The mutant provides the classical four cysteinyl ligation to the [4Fe-4S] cluster. The two proteins were prepared as described previously. 9 The Ga-substituted forms were prepared by reconstitution of the apoproteins 10 essentially following the protocol of Vo et al. 8 The substitution of gallium for iron in metalloproteins is very useful for NMR studies because of the diamagnetic nature of gallium. The coordination chemistry and the ionic radii of gallium and iron are similar, which allows the substitution to occur without dramatic changes in the geometry of the active site of the protein. 8 Ga-substituted [2Fe-2S] Fds have been examined by NMR spectroscopy, but NMR cannot determine metal atom stoichiometry. 8,11 We show herein using Ga-substituted Pf Fd that accurate stoichiometries and structural information can be obtained using the complementary techniques of ESI-FTICR mass spectrometry and NMR. The 500 MHz 1 H NMR spectrum of the diamagnetic Ga- substituted wild-type Pf Fd is compared to the “diamagnetic” 0-10 ppm portion of the NMR spectrum of paramagnetic [3Fe- 4S] Fd A ox (where ox indicates an oxidized cluster and the A form indicates an intact Cys21-Cys48 disulfide bridge) in Figure 1. The strong similarity of the two 1 H NMR spectra is striking and suggests very similar molecular structures. The NMR data are consistent with the presence of either a [4Ga-4S] or a [3Ga-4S] cluster. The apparent increase in the intensity near 8.8 and 7.5 ppm in the peptide NH spectra window and near 1 ppm in the methyl region in diamagnetic Ga-substituted Fd A relative to paramagnetic [3Fe-4S] Fd A ox is due to the suppression by cluster paramagnetism of these resonances in the cluster ligating loop and the turn involving the last cluster ligand in the latter species. Comparison of the TOCSY spectrum of Ga-substituted Fd A (not shown) with that of [3Fe-4S] Fd A ox on the basis of nearly identical NH shifts and TOCSY connectivity readily leads to the assignment * To whom correspondence should be addressed: Department of Bio- chemistry & Molecular Biology, University of Georgia. Phone: 706-542- 2060. Fax: 706-542-0229. E-mail: adams@bmb.uga.edu. ‡ Department of Chemistry, University of Georgia. ² Department of Biochemistry and Molecular Biology, University of Georgia. § University of California. (1) Beinert, H.; Holm, R. H.; Mu ¨nck, E. Science 1997, 277, 653. (2) Conover, R. C.; Kowal, A. T.; Fu, W.; Park, J. B.; Aono, S.; Adams, M. W. W.; Johnson, M. K. J. Biol. Chem. 1990, 265, 8533. (3) Teng, Q.; Zhou, Z.; Smith, E. T.; Busse, S. C.; Howard, J. B.; Adams, M. W. W.; La Mar, G. N. Biochemistry 1994, 33, 6316. (4) Wang, P. L.; Calzolai, L.; Bren, K.L.; Teng. Q.; Jenney, F. E., Jr.; Brereton, P. S.; Howard, J. B.; Adams, M. W. W.; La Mar, G. N. Biochemistry 1999, 38, 8167. (5) (a) Conover, R. C.; Park, J.-B.; Adams, M. W. W.; Johnson, M. K. J. Am. Chem. Soc. 1995, 112, 4562. (b) Fu, W.; Telser, J.; Hoffman. B. M.; Smith. E. T.; Adams, M. W. W.; Johnson, M. K. J. Am. Chem. Soc. 1994, 116, 5722. (c) Finnegan, M. G.; Conover, R. C.; Park, J.-B.; Zhou, Z. H.; Adams, M. W. W.; Johnson, M. K. Inorg. Chem. 1994, 34, 5358. (d) Staples, C. R.; Dhawan, I. K.; Finnegan, M. G.; Dwinell, D. A.; Zhou, Z. H.; Huang, H.; Verhagen, M. F. J. M.; Adams, M. W. W.; Johnson, M. K. Inorg. Chem. 1997, 36, 5740. (6) (a) Sugiura, Y.; Ishizu, K.; Kimura, T. Biochemistry 1975, 14, 97- 101. (b) Bonomi, F.; Ganadu, M. L.; Lubini, G.; Pagani, S. Eur. J. Biochem. 1994, 222, 639. (c) Iametti, S.; Uhlmann, H.; Sala, N.; Bernhardt, R.; Ragg, E.; Bonomi, F. Eur. J. Biochem. 1996, 239, 818. (7) Kazanis, S.; Pochapsky, T. C.; Barnhart, T. M.; Penner-Hahn, J. E.; Mizra, U. A.; Chait, B. T. J. Am. Chem. Soc. 1995, 117, 6625. (8) Vo, E.; Wang, H. C.; Germanas, J. P. J. Am. Chem. Soc. 1997, 119, 1934. (9) (a) Zhou, Z. H.; Adams, M. W. W. Biochemistry 1997, 36, 10892. (b) Brereton, P. S.; Verhagen, M. F. J. M.; Zhou, Z. H.; Adams, M. W. W. Biochemistry 1998, 37, 7351. (10) Pf Fd (60 mg; 10 mL) was denatured with HCl (1 mL; 11.6 M) at 60 °C, collected by centrifugation, and resuspended in 30 mL of Tris‚Cl (0.5 M; pH 8.0). The denaturation steps were repeated three times. The apo-protein was resuspended in 30 mL of Tris‚Cl (0.5 M.; pH 8.0) and DTT (1 mM). Sequentially, Na2S then Ga(NO3)3 (10-fold molar excess of each) were added dropwise with stirring and left overnight at 4 °C. The protein was loaded onto a Pharmacia Q-HP column (2.6 × 10 cm) and washed with 2 column volumes of 50 mM Tris‚Cl (pH 8.0). The Ga-substituted protein was eluted with a gradient (10 column vols) from 0 to 0.6 M NaCI in the same buffer and was concentrated by ultrafiltration (YM-3, Amicon). The sample was applied to a G-75 gel filtration column (3.5 × 60 cm), eluted with 50 mM sodium phosphate, pH 8.0, and concentrated by ultrafiltration. Figure 1. The 0 to 10 ppm portion of the 500 MHz 1 H NMR spectra of (A) wild-type Pf 3Fe FdNA ox and (B) the Ga-substituted FdA (with an intact Cys21-Cys48 bridge). The wild-type Fd exhibits additional strongly relaxed and hyperfine-shifted resonances from the three Cys in the 10- 25 ppm window (data not shown). Selected resolved signals are labeled. 7935 J. Am. Chem. Soc. 2001, 123, 7935-7936 10.1021/ja0160795 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/24/2001