Proton Linkage in Formation of the Cytochrome c-Cytochrome c Peroxidase Complex: Electrostatic Properties of the High- and Low-Affinity Cytochrome Binding Sites on the Peroxidase

Abstract

The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength > or = 100 mM were adequately analyzed in terms of 1:1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< or = 50 mM) and high pH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.

Crystal structure and characterization of a cytochrome c peroxidase–cytochrome c site-specific cross-link

“…c, activity first increases with ionic strength and then decreases. Such complex behavior has been interpreted as an ionic strengthdependent change in the rate-limiting step (30) as well as a multisite model with CCP having both high-affinity and lowaffinity sites (8,(10)(11)(12). As shown in Table 2, the complex is essentially inactive (exhibits Ͻ7.2% activity compared with wild type) toward both substrates at low and high ionic strengths.…”

“…Indeed, several investigations using a variety of methods indicate two cyt. c binding sites (8)(9)(10)(11)(12), although less clear is whether both sites are productive in electron transfer of physiological relevance.…”

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

“…For example, although the affinity of Mn 2ϩ binding to the Mb double variant is Ϸ20-fold greater at the newly created site than at the endogenous metal ion binding site, the affinity of the peroxidase for Mn 2ϩ is about two orders of magnitude greater (29). Interestingly, however, the affinity of the peroxidase for Mn 2ϩ at acidic pH, where catalytic activity of the enzyme is greatest, is the same order of magnitude as that for the variant studied here.…”

“…Binding of Mn 2ϩ ions to wild-type Mb and the variant was studied by monitoring the release of protons upon metal ion binding. Instrumentation (27), preparation of metal ion solutions (28), and data acquisition and analysis procedures (28,29) were reported previously. The dependence of peroxidase activity on pH was evaluated at 25°C between pH 5.0 and 8.0 with a mixed buffer system (5 mM Mes͞5 mM Mops͞5 mM TAPS͞95 mM NaCl) [ (32).…”

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

“…Using recombinant LmP and LmCytc, we found that the steady-state kinetics of the Lm system is far simpler than the yeast system (5). First, the Lm system obeys straightforward Michaelis-Menten kinetics, whereas yeast CCP does not (20) possibly because yeast CCP has a second weak site for Cytc (21,22). Second, LmP exhibits a steady decrease in activity with increasing ionic strength (5), whereas yeast CCP activity increases with ionic strength up to ∼100 mM and then decreases (20).…”