Oxidized and Reduced Azotobacter Vinelandii Ferredoxin I at 1.4 Å Resolution: Conformational Change of Surface Residues without Significant Change in the [3Fe-4S] +/0 Cluster †,‡ C. G. Schipke, D. B. Goodin, D. E. McRee, and C. D. Stout* Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037-1093 ReceiVed December 22, 1998; ReVised Manuscript ReceiVed May 5, 1999 ABSTRACT: The refined structure of reduced Azotobacter Vinelandii 7Fe ferredoxin FdI at 100 K and 1.4 Å resolution is reported, permitting comparison of [3Fe-4S] + and [3Fe-4S] 0 clusters in the same protein at near atomic resolution. The reduced state of the [3Fe-4S] 0 cluster is established by single-crystal EPR following data collection. Redundant structures are refined to establish the reproducibility and accuracy of the results for both oxidation states. The structure of the [4Fe-4S] 2+ cluster in four independently determined FdI structures is the same within the range of derived standard uncertainties, providing an internal control on the experimental methods and the refinement results. The structures of the [3Fe-4S] + and [3Fe-4S] 0 clusters are also the same within experimental error, indicating that the protein may be enforcing an entatic state upon this cluster, facilitating electron-transfer reactions. The structure of the FdI [3Fe-4S] 0 cluster allows direct comparison with the structure of a well-characterized [Fe 3 S 4 ] 0 synthetic analogue compound. The [3Fe-4S] 0 cluster displays significant distortions with respect to the [Fe 3 S 4 ] 0 analogue, further suggesting that the observed [3Fe-4S] +/0 geometry in FdI may represent an entatic state. Comparison of oxidized and reduced FdI reveals conformational changes at the protein surface in response to reduction of the [3Fe-4S] +/0 cluster. The carboxyl group of Asp 15 rotates ∼90°, Lys 84 , a residue hydrogen bonded to Asp 15 , adopts a single conformation, and additional H 2 O molecules become ordered. These structural changes imply a mechanism for H + transfer to the [3Fe-4S] 0 cluster in agreement with electrochemical and spectroscopic results. Iron-sulfur ([Fe-S]) cluster-containing proteins participate in electron transfer, catalytic, regulatory, and structural functions (1-3). Electron-transfer reactions involving [Fe- S] clusters occur in small proteins containing 1, 2, 3, 4, 7, or 8 Fe atoms in one or more clusters or centers in rubredoxins (Rds), 1 ferredoxins (Fds), and high-potential iron proteins (HiPIPs) (2, 4) and in larger multidomain complexes, such as chloroplast cytochrome b 6 f (5). Biologically relevant redox couples occur for Fe 3+/2+ (Sγ) 4 Rd centers, [4Fe- 4S] 3+/2+ clusters in HiPIPs, and [4Fe-4S] 2+/+ and [2Fe- 2S] 2+/+ clusters in Fds; reduction potentials range from -650 mV in [4Fe-4S] 2+/+ Fd clusters to +350 mV in HiPIPs (1). The protein matrix influences cluster reduction potential by controlling solvent accessibility and the number of amide dipoles directed at µ 2 -S, µ 3 -S cluster and Sγ ligand atoms (6). The protein may further influence reduction potential by enforcing an ‘entatic’ or ‘rack’ state, restraining [Fe-S] cluster structure intermediate between ground-state oxidized and reduced geometries (7, 8). In blue copper proteins, for example, this effect raises the reduction potential by enforc- ing distorted tetrahedral geometry on the Cu(I) site and restricting the approach of an axial Met ligand in both the Cu(I) and Cu(II) states; structural reorganization between the oxidized and reduced states is therefore minimized, enhanc- ing the rate of electron transfer (9). Structural characterization of [Fe-S] proteins in both oxidized and reduced states has been limited. In the MoFe protein of nitrogenase, a Ser side chain and a main chain N ligand in the oxidized cluster, P ox , are exchanged for two new Fe-S bonds in the reduced state, P n (10). Concomitant with this structural change, a significant expansion occurs in the P n cluster relative to the P ox cluster as manifested in a 0.15 Å longer average Fe-Fe distance (11). Initial comparison of the structures of Peptococcus aerogenes 8Fe Fd and Chromatium Vinosum HiPIP lead to the observation that protein-bound [4Fe-4S] 2+ clusters can exhibit the [4Fe- 4S] 2+/+ redox couple, as in Fds, or the [4Fe-4S] 3+/2+ couple, as in HiPIPs (12). In C.V. HiPIP the reduced [4Fe-4S] 2+ cluster manifests longer Fe-S bonds (∼0.1 Å), resulting in a tetragonal distortion of the cluster, and shorter (∼0.2 Å) NH‚‚‚S interactions (13). In contrast, oxidized and reduced Pyrococcus furiosus Rd structures refined at 1.8 Å resolution show smaller increases in the Fe-Sγ bond lengths (0.04 Å) † This work supported by the National Institutes of Health (Grant GM48495 to D.B.G. and D.E.M. and Grant GM36325 to C.D.S.). ‡ Coordinates and structure factors for three new, refined ferredoxin structures have been deposited with the Protein Data Bank (Ox2, Red1, and Red2; accession codes 7FD1, 6FDR, and 7FDR, and r7fd1sf, r6fdrsf and r7fdrsf, respectively). * To whom correspondence should be addressed: Phone: 619-784- 8738. Fax: 619-784-2857. E-mail: dave@scripps.edu. 1 Abbreviations: FdI, Azotobacter Vinelandii 7Fe ferredoxin; [2Fe- 2S], [3Fe-4S], and [4Fe-4S], protein-bound [Fe-S] clusters; [Fe2S2], [Fe3S4], and [Fe4S4], core structures in synthetic [Fe-S] cluster analogue compounds; Rd, rubredoxin; Fd, ferredoxin; HiPIP, high-potential iron protein; su, standard uncertainty; EPR, electron paramagnetic resonance. 8228 Biochemistry 1999, 38, 8228-8239 10.1021/bi983008i CCC: $18.00 © 1999 American Chemical Society Published on Web 06/10/1999