The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b(5)

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b(5), three mutants at a highly conserved residue V a145, which is a member of heme hydrophobic pocket residues have been chara cterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were construc ted to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyro sine still sitting inside the hydrophobic pocket, while the side chain of t he mutant V45E and V45H shift away from the heme pocket. The redox potentia ls of mutants V45Y, V45H, V45E and wild-type of cytochrome b(5) are -35 mV, 8 mV, - 26 mV and - 3 mV, respectively. The bigger change of the V45Y on r edox potential is due to the close contact between the hydroxyl group and t he heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stab ility of these mutants towards heat have been observed with the order: WT > V45Y similar to V45H > V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT > V45Y > V45H > V45E. These results suggest that the difference in the h ydrophobic free energy is a major factor contributing to the stability of t he Va145 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Va145 plays an import ant role in the stability and redox potential of the protein. (C) 2000 Else vier Science B.V. All rights reserved.