Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry

Abstract

Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN 3 with similar affinities at 25 °C. The pH-independent equilibrium association constants for formation of the CcP · HN 3 and Mb · HN 3 complexes are (1.05 ± 0.06) × 10 5 and (1.6 ± 0.8) × 10 5 M −1, respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The Δ H 0 values for formation of CcP · HN 3 and Mb · HN 3 are −16.4 ± 0.7 and −9.0 ± 0.5 kcal/mol, respectively, and the Δ S 0 values are −32 ± 2 and −16 ± 2 cal/deg mol, respectively. The proton associated with HN 3 is retained in both protein complexes at low pH but dissociates with apparent p K A values of 5.5 ± 0.2 and ⩾8.2 for the Mb · HN 3 and CcP · HN 3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N 3 −. CcP binds N 3 − very weakly, if at all, and only an upper-limit of 18 ± 5 M −1 for the pH-independent equilibrium association constant for the CcP · N 3 − complex can be determined. Mb binds N 3 − with an association constant of (1.8 ± 0.1) × 10 4 M −1. The ratio of the equilibrium association constants for HN 3 and N 3 − binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN 3. Protonation of His-64 in Mb enhances HN 3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN 3 due to loss of base-assisted association of the ligand to the heme iron.