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 °...