Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase

Peroxidases typically bind their reducing substrates weakly, with K-d value s in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferr ic horseradish peroxidase isoenzyme C (HRPC) [K-d = 2.4 muM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provi ded a useful tool for probing the environment of the peroxidase aromatic-do nor-binding site and the distal heme cavity. Knowledge of the underlying th ermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isotherma l titration calorimetry results of this study on the binding of aromatic hy droxamic acid analogues to ferric HRPC under nonturnover conditions (no H2O 2 present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to H RPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. T he overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, w ith the distal Ar38 acting as an "entropic gate keeper". Dramatic enthalpy- entropy compensation is observed for BHA and 2-naphthohydroxamic acid bindi ng to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due t o increased flexibility of Arg38 in the complexes at low pH and greater acc ess by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the Kd values corrected for substrate ionization suggests that enthalpy-entropy compensation is operati ve over a wide pH range. Examination of the thermodynamics of binding of ri ng-substituted hyrazides to HRPC reveals that the binding affinities of aro matic donors are highly sensitive to the position and nature of the ring su bstituent.