Molecular Structure of the Oxidized High-Potential Iron-Sulfur Protein Isolated from Ectothiorhodospira vacuolataT9t

The high-potential iron-sulfur protein (iso-form 11) isolated from Ectothiorhodospira vacuolata has been crystallized and its three-dimensional structure determined by molecular replacement procedures and refined to 1.8-A resolution with a crystallographic R factor of 16.3%. Crystals employed in the investigation belonged to the space group C222, with unit cell dimensions of a = 58.4 A, b = 64.7 A, and c = 39.3 A and one molecule per asymmetric unit. Like those HiPIPs structurally characterized thus far, the E. vacuolata molecule contains mostly reverse turns that wrap around the iron-sulfur cluster with cysteine residues 34,37,5 1, and 65 ligating the metal center to the polypeptide chain. There are 57 ordered solvent molecules, most of which lie a t the surface of the protein. Two of these water molecules play important structural roles by stabilizing the loops located between Asp 42 and Lys 57. The metal center binding pocket is decidedly hydrophobic with the closest solvent molecule being 6.9 A from S2 of the [4Fe-4S] cluster. The E. vacuolata HiPIP molecules pack in the crystalline lattice as dimers with their iron-sulfur centers approximately 17.5 A apart. On the basis of biochemical properties, it was anticipated that the E. vacuolata HiPIP would be structurally more similar to the HiPIP isolated from Ectothiorhodospira halophila than to the protein obtained from Chromatium vinosum. In fact, the E. vacuolata molecule is as structurally close to the C. vinosum HiPIP as it is to the E. halophila protein due to the presence of various insertions and deletions that disrupt local folding. The E. vacuolata HiPIP structure thus calls into question whether molecular modeling experiments, based on primary structure homology alone, are valid when numerous insertions and deletions are present. The high-potential iron-sulfur proteins (HiPIPs) are a group of low molecular weight electron transport proteins typically isolated from purple phototrophic bacteria (Bartsch, 1978). A key feature of these proteins is the presence of a [4Fe-4S] cluster which undergoes a reversible one-electron transfer reaction at a characteristically high oxidation-reduction midpoint potential of between +50 and +450 mV (Meyer et al., 1983). The first three-dimensional structure of a HiPIP, namely, that isolated from Chromatium vinosum, was determined nearly 20 years ago in the laboratory of Dr. Joseph Kraut (Carter et al., 1974; Freer et al., 1975). From that elegant X-ray investigation, it was shown that the secondary structure of the C. vinosum HiPIP consists of two short a-helical segments, three strands of antiparallel @-pleated sheet, and various reverse turns that wrap around to bury the [4Fe-4S] center within the protein matrix. In addition, the X-ray analysis demonstrated that the irons and inorganic sulfurs of the prosthetic group adopt a cubane-type configuration as also observed in the three-dimensional structure of the low-potential bacterial ferredoxin isolated from Peptococcus aerogenes (Adman et al., 1973, 1976). Unlike the ‘This research was supported in part by grants from the NIH (GM30982 to H.M.H. and GM21277 to T.E.M.). H.M.H. is an Established Investigator of the American Heart Association. * X-ray coordinates for the E. vacuolata HiPIP have been deposited in the Brookhaven Protein Data Bank (accession no. 1HPI; (Bernstein etal., 1977) ormaybeobtainedimmediatelyviaHOLDEN@ENZYME. WISC.EDU. * To whom correspondence should be addressed. 8 University of Wisconsin-Madison. 11 University of Arizona. @ Abstract published in Advance ACS Abstracts, February 15, 1994. HiPIPs, however, the low-potential ferredoxins contain two such clusters per polypeptide chain and transfer electrons at low redox potentials near -400 mV (Stombaugh et al., 1976). When the three-dimensional structures of the reduced C. vinosum HiPIP and the oxidized P. aerogenes ferredoxin were subsequently compared, the iron-sulfur clusters appeared to be indistinguishable (Carter et al., 1972). From this comparison, the so-called “three-state” hypothesis was set forth to reconcile the vastly differing magnetic properties and redox potentials displayed by these two proteins (Carter et al., 1972). The main premise of the theory was that three oxidation states are available to the [4Fe-4S] cluster with overall net charges of -1, -2, and -3 when attached to a protein via cysteinyl ligands. The HiPIPs transfer electrons between the -1 and -2 states, whereas the low-potential ferredoxins utilize the -2 and -3 states. The HiPIPs and ferredoxins thus share in common the -2 state which is EPR silent. Prior to the proposal of Carter et al. (1972), the synthetic analog ( E ~ ~ N ) ~ [ F ~ ~ S ~ ( S C H Z P ~ ) ~ ] was prepared and chemically characterized, and its X-ray structure was determined by Herskovitz et al. (1972). On the basis of proton magnetic resonance, Mossbauer, photoelectron, and electronic spectra, and magnetic susceptibility, the authors concluded that the oxidation states of the synthetic analog, the reduced form of the HiPIP, and the oxidized form the low-potential bacterial ferredoxin were equivalent, thus lending further evidence in support of the “three-state” hypothesis. The factors that determine which oxidation states are available to the metal center when associated with a protein are still being investigated and are subject to much speculation. It has been suggested, for example, that theextent of hydrogen 0006-2960/94/0433-2476$04.50/0