Proton NMR characterization of the catalytically relevant proximal and distal hydrogen-bonding networks in ligated resting state horseradish peroxidase

Abstract

Proton NMR spectroscopy of the low-spin cyanide complex of horseradish peroxidase, HRPCN, in H20 solution was used to examine exchangeable resonances of functionally important amino acids in the heme pocket and their role in hydrogen-bonding networks, which have been proposed to facilitate peroxidase catalysis. Spectra were analyzed by use of nuclear Overhauser effect and saturation-transfer spectroscopies, along with consideration of paramagnetic shift and relaxation.Definitive assignments could be made in spite of the size of the proteins (42 kDa), its inherent paramagnetism (iron(III), 5 = */2), and the relatively few resolved resonances, suggesting that these NMR methods may be applicable to even larger heme proteins. The resonance identification was made for labile protons of the proximal His-170, distal His-42, and a heme peripheral contact, Tyr-185, which confirm the close similarity of the heme pocket stereochemistry of horseradish and cytochrome c peroxidases. The resonance assignments enabled determination of several key facets of the structure of the heme cavity of HRPCN. The proton taken up in concert with anionic ligand binding is shown to be present on the His-42 imidazole ring, forming a imidazolium side chain that hydrogen bonds sufficiently strongly to the nitrogen of the bound cyanide to induce a detectable isotope effect on the electronic structure of the heme. The strength of the hydrogen bond is confirmed by the stability of this network over the pH range 4-11. The labile ring proton of the proximal His-170 in HRPCN was located in a position that is consistent with an imidazolate description of the axial ligand but with the proton transferred to a nearby amino acid acceptor site less than 1 Á removed. The structural changes in the heme and proximal and distal amino acid residues upon ligand binding is discussed as a model for HRP compound II formation.

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

“…The resolved upfield spectral region displays several single-proton resonances and a few multiproton signals. Previous work on both heme model compounds and a number of heme peroxidases have firmly concluded that this spectral region encompasses the resonances from ␤-protons of the heme vinyl and propionate groups as well as those from some of the amino acid residues near the heme center (53,72,(77)(78)(79). The chemical shifts and the corresponding diamagnetic shift values predicted from Curie plot as well as the spin-lattice relaxation times for the hyperfine shifted resonances and their assignments in CPOCN are compiled in Table I, along with the corresponding parameters in MnPCN (72,80) and HRPCN (51) reported previously.…”

“…This is anticipated, since the distal acid-base catalyst in CPO is a glutamic acid (6) rather than a histidine as in other heme peroxidases (24, 74 -76). The proton/deuteron exchange on the N⑀ atom of the distal histidine has been shown to be responsible for the observed solvent effect on heme resonances in several heme peroxidases (36,53,72).…”

“…The assignment of the hyperfine shifted signals for the CPOCN complex was achieved through comparison with the assignments made for cyanide derivatives of other heme peroxidases (30,34,36,53,72) and examination of the active site structure of CPO revealed by its crystal structure (6) with confirmation through one-and two-dimensional NOE measurements as well as through bond connectivities (COSY). Shown in S1) connectivities prove the validity of previous NOE connectivities observed in one-dimensional NOE experiments (54) and lead to the proposed assignments for all but three of the nonexchangeable hyperfine shifted protons (resonances A, B, and Z) from the heme active site in CPOCN.…”

“…Compared with the extensive and in depth NMR studies on CcP (28 -36) and horseradish peroxidase (34,(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53), relatively few NMR studies of CPO have been reported (54 -58). Furthermore, the most powerful NMR approach, the two-dimensional NMR technique that has led to the unambiguous assignment of major hyperfine-shifted signals in a number of heme peroxidases (59), has not been applied to the investigation of CPO.…”

Application of the paramagnetic dipole field for solution NMR active site structure determination in low‐spin, cyanide‐inhibited ferric hemoproteins

“…Yeast cytochrome c peroxidase, CcP, yielded the first crystal structure (37), while horseradish peroxidase, HRP, because of its much greater stability, yielded the more detailed NMR assignment and solution NMR characterization (1,15,38,39). The cyanide-inhibited complex mimics the catalytically active compound-II of heme peroxidases (40).…”