THE STRUCTURE AND FUNCTION OF OMEGA-LOOP-A REPLACEMENTS IN CYTOCHROME-C

The structural and functional consequences of replacing OMEGA-loop A ( residues 18-32) in yeast iso-1-cytochrome c with the corresponding loo p of Rhodospirillum rubrum cytochrome c2 have been examined. The three -dimensional structure of this loop replacement mutant RepA2 cytochrom e c, and a second mutant RepA2(Val 20) cytochrome c in which residue 2 0 was back substituted to valine, were determined using X-ray diffract ion techniques. A change in the molecular packing is evident in the Re pA2 mutant protein, which has a phenylalanine at position 20, a residu e considerably larger than the valine found in wild-type yeast iso-1-c ytochrome c. The side chain of Phe 20 is redirected toward the molecul ar surface, altering the packing of this region of OMEGA-loop A with t he hydrophobic core of the protein. In the RepA2(Val 20) structure, OM EGA-loop A contains a valine at position 20, which restores the origin al wild-type packing arrangement of the hydrophobic core. Also, as a r esult of OMEGA-loop A replacement, residue 26 is changed from a histid ine to asparagine, which results in displacements of the main-chain at oms near residue 44 to which residue 26 is hydrogen bonded. In vivo st udies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2 (Val 20) cytochrome c behaves much like the w ild-type yeast iso-1 protein, whereas the stability and function of th e RepA2 cytochrome c showed a temperature dependence. The midpoint red uction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25-degrees-C. This is 19 mV less than the wild-type and Rep A2(Val 20) proteins (290 mV) and may result from disruption of the hyd rophobic packing in the heme pocket and increased mobility of OMEGA-lo op A in RepA2 cytochrome c. The temperature dependence of the reductio n potential is also greatly enhanced in the RepA2 protein.