Effects of surface amino acid replacements in cytochrome c peroxidase on complex formation with cytochrome c

Abstract

Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase. The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins. The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography. The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme. The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase. The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c. This empirical finding localizes a primary recognition region critical to the dynamic complex. Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.

An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule

“…In the case of a direct titration experiment, this causes little problem because the corresponding equation is only quadratic [4,5], and the physically meaningful root can be easily identified. In the case of a competitive displacement experiment, however, the corresponding equation becomes cubic and thus one would be faced with the possible existence of multiple roots [8,12]. A similar problem was also encountered in the determination of a dissociation constant for the recepto~ non-radioactive ligand complex from displacement curves [7].…”

“…However, it is not easy to extend the analytical, algebraically explicit description of binding equilibria to mixtures of competing ligands. For a system consisting of a protein and two competing ligands, when the total concentrations of all the components are of the same order of magnitude, combining all partial equilibria and mass conservation, one obtains a cubic algebraic equation [7,8]. Roots of polynomials degree n > 2 are usually extracted by methods of numerical analysis …”

Interaction Domain for the Reaction of Cytochrome c with the Radical and the Oxyferryl Heme in Cytochrome c Peroxidase Compound I

“…The shielding effect on residues 221 and 224 must therefore arise from multiple binding sites, or long-range conformational effects. Corin et al (1991Corin et al ( , 1993 have found that substitution of Asp-79 or Asp-217 with Lys led to only relatively subtle effects on binding, steady-state electron transfer activity, and intracomplex electron transfer, indicating that these residues are probably not directly involved in the interaction domain. However, substitution of Asp-37 with Lys reduced both the binding strength and the steadystate electron transfer activity more than 10-fold.…”

“…These results supported the general features of the Poulos-Kraut model, except that Asp-79 of CcP did not appear to be involved in complex formation (Bechtold & Bosshard, 1985). More recently, Corin et al (1991Corin et al ( ,1993 and Hake et al (1992) found that replacement of Asp-37 on CcP with a lysine residue greatly decreased both the binding affinity and the steady-state electron transfer activity, as predicted by the Poulos-Kraut model. However, replacement of Asp-79 or Asp-217 with a lysine residue had only very subtle effects on binding and electron transfer activity, suggesting that these residues arenot directly involved in the interaction.…”

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

“…A number of other studies using computer modeling (15-17) and chemical modification (18) have identified additional residues on CcP that may be important in cytochrome c binding. These include Asp-33, Asp-37, Asp-79, and residues between positions 221-224 and 290-294 as potential interaction sites.Site-directed mutagenesis of CcP has been used to test some of the suggested locations for the cytochrome c binding sites (21)(22)(23)(24)(25)(26)(27). These studies consistently find that charge-neutralization or charge-reversal mutants of Asp-34, Asp-37, and Glu-290 inhibit cytochrome c binding while mutations at Glu-32, Glu-35, Asp-79, Asp-148, Asp-217, and Glu-291 have little effect on cytochrome c binding.…”