MOLECULAR-STRUCTURE OF THE OXIDIZED HIGH-POTENTIAL IRON-SULFUR PROTEIN ISOLATED FROM ECTOTHIORHODOSPIRA-VACUOLATA

The high-potential iron-sulfur protein (iso-form II) isolated from Ectothiorhodospira vacuolata has been crystallized and its three-dimensional structure determined by molecular replacement procedures and refined to 1.8-Angstrom resolution with a crystallographic R factor of 16.3%. Crystals employed in the investigation belonged to the space group C222(1) with unit cell dimensions of alpha = 58.4 Angstrom, b 64.7 Angstrom and 39.3 Angstrom 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, 51, and 65 ligating the metal center to the polypeptide chain. There are 57 ordered solvent molecules, most of which lie at 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 Angstrom 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 Angstrom 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.