3-DIMENSIONAL STRUCTURE OF CYTOCHROME-C' FROM 2 ALCALIGENES SPECIES AND THE IMPLICATIONS FOR 4-HELIX BUNDLE STRUCTURES

The three-dimensional structures of two cytochromes c' have been deter mined in order to analyse the common features of proteins of this fami ly and their relationship with other four-helix bundle structures. The structure of cytochrome c' from Alcaligenes sp was determined by mole cular replacement supplemented with the iron anomalous scattering and the use of a single isomorphous heavy-atom derivative, and was refined using synchrotron data to 1.8 Angstrom resolution. The final model, c omprising 956 protein atoms (one monomer) and 89 water molecules, has a final R value of 0.188 for all data in the range 20.0-1.8 Angstrom r esolution (14 673 reflections). The structure of the cytochrome c' fro m Alcaligenes denitrificans is isomorphous and essentially identical ( r.m.s. deviation for all atoms 0.36 Angstrom). Although its amino-acid sequence has not been determined chemically, only four differences fr om that of Alcaligenes sp cytochrome c' were identified by the X-ray a nalysis. The final model for Alcaligenes denitrificans cytochrome c', comprising 953 protein atoms and 75 water molecules, gave a final R fa ctor of 0.167 for all data in the range 20.0-2.15 Angstrom (8220 refle ctions). The cytochrome cl monomer forms a classic four-helix bundle, determined by the packing of hydrophobic side chains around the enclos ed haem group. There are very few cross-linking hydrogen bonds between the helices, the principal sidechain hydrogen bonding involving one o f the haem propionates and a conserved Arg residue. The cytochrome c' dimer is created by a crystallographic twofold axis. Monomer-monomer c ontacts primarily involve the two A helices, with size complementarity of side chains in a central solvent-excluded portion of the interface and hydrogen bonding at the periphery. Both species have a pyroglutam ic acid N-terminal residue. The haem iron is five-coordinate, 0.32 Ang strom out of the haem plane towards the fifth ligand, His120. The unus ual magnetic properties of the Fe atom may be linked to a conserved ba sic residue, Arg124, adjacent to His120.