Structure, interaction and electron transfer between cytochrome b(5), its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fr agment of bovine liver cytochrome b(5) in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical ti tration tin the presence of 1 mM (Ru(NH3)(6))(3+), pH 7.0 and I = 0.1 M) ar e 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), respe ctively. 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 th at 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 toward s 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 mo de between Glu44 and Glu56 with cytochrome c. The binding between cytochrom e c and cytochrome b(5) was studied by optical difference spectra of cytoch rome 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 cyt ochrome 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 formati on between cytochrome b(5) and cytochrome c. The reduction of horse heart f erricytochrome c by recombinant ferrocytochrome b(5) and its mutants has be en studied. The rate constant of the electron transfer reaction between fer ricytochrome 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 r ate constants are strongly dependent on ionic strength and temperature. The se studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b, and cytochrome c is electrostati cally guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction. (C) 1999 Academic Press.