H-1-NMR INVESTIGATION OF THE INFLUENCE OF INTERACTING SITES ON THE DYNAMICS AND THERMODYNAMICS OF SUBSTRATE AND LIGAND-BINDING TO HORSERADISH-PEROXIDASE

The influence of substrate benzhydroxamic acid (BHA) and iron ligand (cyanide) on the thermodynamics and dynamics of each of the two binding sites of horseradish peroxidase (HRP) isozyme C has been investigated by H-1 NMR spectroscopy. A combination of line-width analysis and saturation transfer spectroscopy has allowed the direct determination of the off-rate of substrate and ligand in the absence or presence of the other. These off-rates, together with available dissociation constants obtained by optical spectroscopy (Schonbaum, 1973), provide estimates for k(on). The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the H-1 NMR determined dynamic and thermodynamic data agree well with those values available in the literature. BHA binding leads to a 200-fold decrease in CN- affinity that arises from a factor >10 decrease in k(off)(CN-) and >2 x 10(3) decrease in k(on)(CN-). While a portion of the decrease in k(on)(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in k(on)(CN-) and that in k(off)(CN-) indicates that BHA in the binding pocket blocks the CN- ligation channel and serves as a "gate" to CN- exchange. This view is supported by observing a factor >4 decrease in distal His labile proton exchange with bulk water in HRP-CN upon BHA binding. The ternary complex BHA.HRP-CN is shown to be heterogeneous. While the thermodynamics of BHA and CN- binding appear similar in the two ternary complexes, the BHA on- and off-rates for the two complexes differ by a factor of approximately 10. The two heterogeneous forms interconvert at 25-degrees-C at approximately 2 x 10(2) s-1, precluding the determination of any difference in the CN- binding rates by saturation transfer. The greater lability of one of the two ternary complexes is attributed to an alternate orientation of some distal residue that blocks the substrate binding channel in one of the forms. Transferred nuclear Overhauser effects from the heme to BHA in the ternary complex reveal that the BHA substrate is in contact not only with the heme pyrrole D substituents but also with the distal His 42, indicating that the polar side chain of BHA extends well into the distal heme pocket. Hence it does not appear that heme peroxidase substrates need to be sterically blocked from the iron binding site to account for the failure to transfer the ferryl oxo group to most substrates. A model for the binding of BHA to HRP is presented where the hydroxamine acid proton hydrogen bonds to the distal His 42. This model accounts for the unique difference in binding constants to HRP and HRP-CN for BHA relative to other substrates and suggests a structural basis for the optimal role of aromatic peracids in activating HRP.