IMPORTANCE OF A CONSERVED PHENYLALANINE-35 OF CYTOCHROME B(5) TO THE PROTEINS STABILITY AND REDOX POTENTIAL

Phenylalanine-35, which is a residue of the hydrophobic patch on the s urface of cytochrome b(5), has been mutated into Tyr35, His35 and Leu3 5 to elucidate the functions of the Phe35 and give further insight int o the roles of the hydrophobic patch and/or aromatic network, The effe cts of these mutations on the heme environment, denaturation towards h eating and the denaturant urea, redox potential and stability of prote in were studied, The relative stability of cytochrome b(5) and its mut ants towards heating has the order Phe35Tyr > wild type > Phe35Leu > P he35His in the oxidized state and wild type > Phe35Tyr > Phe35Leu > Ph e35His in the reduced state, All the mutants exhibit decreased reducti on potentials: Phe35Tyr -66 mV, Phe35His -51 mV and Phe35Leu -28 mV, w hich are more negative than that of the wild type. The order of redox potential reflects the relative stability in the oxidized and reduced states, A method of producing multiple mutants at a single site of a g ene is also described for the first time.