Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bovine liver cytochrome b(5) in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration tin the presence of 1 mM (Ru(NH3)(6))(3+), pH 7.0 and I = 0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b(5), respectively. A comparative two-dimensional NMR study of cytochrome b(5) and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42 similar to Glu44 segment in cytochrome b(5) may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b(5) was studied by optical difference spectra of cytochrome c and variants of cytochrome b(5). The association constants (K-A) for the wild-type, E44A, E56A, and E44/56A mutants of cytochrome b(5) with cytochrome c, are 4.70(+/- 0.10) x 10(6) M-1, 1.88(+/- 0.03) x 10(6) M-1, 2.70(+/- 0.13) x 10(6) M-1, and 1.14(+/- 0.05) x 10(6) M-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b(5) and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b(5) and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrome b(5) (1.074(+/- 0.49) x 10(7) M-1 s(-1)) is higher than those of the mutant protein E44A (8.98(+/- 0.20) x 10(6) M-1 s(-1)), E56A (8.76(+/- 0.39) x 10(6) M-1 s(-1)), and E44/56A (8.02(+/- 0.38) x 10(6) M-1 s(-1)) at 15 degrees C, pH 7.0, I = 0.35 M. The rate constants are strongly dependent on ionic strength and temperature. These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b, and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction. (C) 1999 Academic Press.