Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.

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 1H 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 kon. The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the 1H 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 greater than 10 decrease in koff(CN-) and greater than 2 x 10(3) decrease in kon(CN-). While a portion of the decrease in kon(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in kon(CN-) and that in koff(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 greater than 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.(ABSTRACT TRUNCATED AT 400 WORDS)

The cyanide-inhibited complexes of two horseradish peroxidase acidic isozymes, A1 (HRPA1, unsequenced) and A2 (HRPA2, sequenced), have been examined by solution two-dimensional 1H NMR methods, and the active site molecular and electronic structure compared to that of the well-characterized isozyme C (HRPC) (Chen, Z., de Ropp, J.S., Hernández, G., & La Mar, G.N. (1994) J. Am. Chem. Soc. 116, 8772-8783), as well as to that of cytochrome c peroxidase. The identity and alignment of catalytically relevant residues near the active site for HRPA1-CN and HRPA2-CN are determined, and key residue replacements implicated in the differential catalytic properties of the acidic vs C isozymes are identified. Heme and axial His contact shift patterns, as well as dipolar contacts of residues with the heme and with each other, confirm a highly conserved structure among the three isozymes, including for the distal pocket residues involved in the activation of the enzyme. The remarkable dynamic stability of the heme pocket, as reflected in NH exchange with solvent, is also conserved for the three isozymes. An additional heme contact, Ile 148, is identified in HRPC-CN. Four residues in contact with the heme in HRPC-CN are replaced in HRPA2-CN, two of which are likely functionally neutral, Gly 169-->Ala and Ile 148-->Leu. However, two substitutions in the acidic isozymes in the aromatic substrate binding pocket on the heme edge, Ile 244-->Leu and Phe 179 or 221-->aliphatic residue, could well account for the dramatic decrease (approximately 10(3) in aromatic substrate binding in the A1 and A2 isozymes vs the C isozyme of HRP.(ABSTRACT TRUNCATED AT 250 WORDS)