Effects of some charged amino acid mutations on the electron self-exchangekinetics of cytochrome b(5)

Two mutants of trypsin-solubilized bovine liver cytochrome bs (cyt bs) have been prepared to elucidate the function of charged residues near the heme exposed edge in the electron self-exchange kinetics of cyt bs. The Glu44, G lu56, and Asp60 were mutated into alanines in mutant I (E44A/E56A/D60A) whi le Glu44, Glu48, Glu56, and Asp60 were mutated into alanines in mutant II ( E44A/E48A/E56A/D60A). The electron self-exchange rates of cyt b(5) mutants I and II have been measured as a function of temperature and ionic strength using the one-dimensional H-1 NMR saturation transfer method. Under the co ndition of mu = 0.10 M and [cyt b(5)] = 0.50 mM, the rate constant of the c yt b(5) mutant I is (4.0 +/- 0.4) x 10(3) M-1 s(-1) st 20 degrees C. The va lue rises to (7.2 +/- 0.7) x 103 M-1 s(-1) at 35 degrees C; data from 20 to 35 degrees C gave Delta H double dagger = 6.8 +/- 0.8 kcal mol(-1) and Del ta S double dagger = -19 +/- 3 eu. The rate constant for cyt b(5) mutant II is (8.4 +/- 0.7) x 103 M-1 s(-1) at 20 degrees C with mu = 0.10 M and [cyt b(5)] = 0.60 mM. The value rises to (17 +/- 1) x 10(3) M-1 s(-1) at 35 deg rees C, Delta H double dagger = 7.9 +/- 0.9 kcal mol(-1), and Delta S doubl e dagger = -14 +/- 4 eu. The rate constant for cyt b(5) mutants increases w ith increasing of ionic strength. The values of the rate constants of cyt b (5) mutants extrapolated to infinite ionic strength, k(inf), are 2.8 x 10(5 ) and 1.1 x 10(5) M-1 s(-1) for mutant I and mutant II, respectively. The p rotein dipole moment projections through the heme exposed edge for two muta nts were obtained from a fit of ionic strength dependence of self-exchange rate constants using van Leeuwen's approach. The dipole moment projections were -220 D and -254 D for oxidized and reduced forms of cyt b(5) mutant I, and -134 D (oxidized) and -159 D (reduced) for cyt b(5) mutant II. In term s of Marcus theory, the reorganization energies of cyt b(5) mutant I and mu tant II have been estimated to be 1.2 and 1.3 eV, respectively. The associa tion constants for the electron self-exchange reactions of mutant I and mut ant II are 0.33 and 1.5 M-1, respectively. Based on above results, the reco gnition and electron self-exchange mechanism between oxidized and reduced s tates of cytochrome b(5) was discussed.