Effects of some charged amino acid mutations on the electron self-exchange kinetics of cytochrome b5

Abstract

Two mutants of trypsin-solubilized bovine liver cytochrome b5 (cyt b5) have been prepared to elucidate the function of charged residues near the heme exposed edge in the electron self-exchange kinetics of cyt b5. The Glu44, Glu56, and Asp60 were mutated into alanines in mutant I (E44A/E56A/D60A) while Glu44, Glu48, Glu56, and Asp60 were mutated into alanines in mutant II (E44A/E48A/E56A/D60A). The electron self-exchange rates of cyt b5 mutants I and II have been measured as a function of temperature and ionic strength using the one-dimensional 1H NMR saturation transfer method. Under the condition of μ = 0.10 M and [cyt b5] = 0.50 mM, the rate constant of the cyt b5 mutant I is (4.0 ± 0.4) × 103 M-1 s-1 at 20°C. The value rises to (7.2 ± 0.7) × 103 M-1 s-1 at 35°C; data from 20 to 35°C gave ΔH‡ = 6.8 ± 0.8 kcal mol-1 and ΔS‡ = -19 ± 3 eu. The rate constant for cyt b5 mutant II is (8.4 ± 0.7) × 103 M-1 s-1 at 20°C with μ = 0.10 M and [cyt b5] = 0.60 mM. The value rises to (17 ± 1) × 103 M-1 s-1 at 35°C, ΔH‡ = 7.9 ± 0.9 kcal mol-1, and ΔS‡ = -14 ± 4 eu. The rate constant for cyt b5 mutants increases with increasing of ionic strength. The values of the rate constants of cyt b5 mutants extrapolated to infinite ionic strength, kinf, are 2.8 × 105 and 1.1 × 105 M-1 s-1 for mutant I and mutant II, respectively. The protein dipole moment projections through the heme exposed edge for two mutants 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 b5 mutant I, and - 134 D (oxidized) and -159 D (reduced) for cyt b5 mutant II. In terms of Marcus theory, the reorganization energies of cyt b5 mutant I and mutant II have been estimated to be 1.2 and 1.3 eV, respectively. The association constants for the electron self-exchange reactions of mutant I and mutant II are 0.33 and 1.5 M-1, respectively. Based on above results, the recognition and electron self-exchange mechanism between oxidized and reduced states of cytochrome b5 was discussed. © 1999 American Chemical Society.