3-DIMENSIONAL STRUCTURE OF FERRICYTOCHROME-C' FROM RHODOSPIRILLUM-RUBRUM AT 2.8 A RESOLUTION

The structure of ferricytochrome c' extracted from Rhodospirillum rubrum has been determined by the X-ray crystallographic method. Crystals in hexagonal space group P6(1), with unit-cell dimensions a = b = 51.72 angstrom and c = 155.49 angstrom, contain one dimer molecule composed of chemically identical polypeptide chains (monomer I and monomer II) per asymmetric unit. An electron density map has been calculated at a resolution of 2.8 angstrom by the multiple isomorphous replacement method using four-circle diffractometer data from native crystals and two heavy-atom derivatives. The quality of the map was improved by averaging the electron density about the non-crystallographic 2-fold axis relating the two monomers. The initial three-dimensional model of monomer I was built on a computer graphics system and that of monomer II was derived from monomer I using the non-crystallographic symmetry matrices. The dimer structure has been refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The current model includes 244 amino acid residues (122 x 2) and 2 hemes, with a root-mean-square deviation in bond lengths from ideal values of 0.022 angstrom. The current crystallographic R-factor is 23.3% for 4,481 independent reflections [\F(o)\ greater-than-or-equal-to sigma(F)] between 5.0 and 2.8 angstrom resolution. The monomer molecule is structurally organized as an array of four nearly parallel alpha-helices which construct a left-twisted bundle. One end of the bundle, in which a covalently bound protoheme IX prosthetic group is incorporated, is more divergent than the other. These structural features of the main-chain folding are very similar to those of the ferricytochrome c' from Rhodospirillum molischianum, although the homology of the amino acid sequences between them is only 24%. The two monomers are assembled to give a dimer through a local 2-fold symmetry, where the interactions are mainly composed of hydrogen bonds between hydrophilic side-chains from the corresponding alpha-helices of the two monomers related by the non-crystallographic 2-fold symmetry, in contrast to hydrophobic interactions of R. molischianum cytochrome c'. The heme-iron is coordinated by the four pyrrole nitrogen ligands in the porphyrin plane and the single axial ligand provided by the N-epsilon-2 atom of His120. The sixth axial ligand site is not occupied by a possible candidate for a ligand, but the side-chain of Leu14 is the closest to the site. The heme iron is displaced 0.5 angstrom from the mean pyrrole nitrogen plane toward the coordinating histidine imidazole ligand.