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We have measured hyperfine shifted NMR signals associated with deuterated imidazole bound to the high-spin Fe3+ state of cytochrome c peroxidase using deuterium magic angle spinning solid-state NMR. These experiments were performed on a mutant of cytochrome c peroxidase, CcP(H175G), for which replacement of the proximal histidine with glycine produced a cavity that can bind a variety of substituted imidazoles including imidazole or 2-methylimidazole. The mutant with imidazole bound is inactive; specifically its reaction with H2O2 is blocked. We observed deuterium NMR signals from the methyl-d3 group of the perdeuterated 2-MeIm sample bound to H175G CcP. The signal displayed an upfield chemical shift and exhibited non-Curie temperature dependence, indicating the existence of low-lying excited electronic states. Upon introducing a nondeuterated competitive ligand, imidazole, a decrease in the intensity of this signal was detected, consistent with the assignment of the deuterium signal to the bound 2-methyli...

Conformational changes that gate the access of substrates or ligands to an active site are important features of enzyme function. In this report, we describe an unusual example of a structural rearrangement near a buried artificial cavity in cytochrome c peroxidase that occurs on binding protonated benzimidazole. A hinged main-chain rotation at two residues (Pro 190 and Asn 195) results in a surface loop rearrangement that opens a large solvent-accessible channel for the entry of ligands to an otherwise inaccessible binding site. The trapping of this alternate conformational state provides a unique view of the extent to which protein dynamics can allow small molecule penetration into buried protein cavities.

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.

The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.

In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.

Site-directed mutagenesis offers a powerful probe of heme proteins. Recent attention has focused on mutants whose proximal iron ligand is replaced by smaller, noncoordinating amino acids. These mutants form artificial cavities where the native proximal ligand resided and which are capable of binding exogenous ligands such as imidazoles. Such reconstituted systems permit detailed studies of the electronic and stereochemical influences of the proximal ligand on catalysis and activity.1-6 The paradigm of these mutants is H175G cytochrome c peroxidase (CCP). Its X-ray crystal structure revealed that the iron is close to two water molecules.1 In heme oxygenase, the H25A mutant has been proposed to contain a single water ligand on the heme iron.4 The iron in the H170A mutant of horseradish peroxidase is probably bound by a water molecule at pH 4 and an additional sixth ligand (possibly a distal histidine) at pH 5-7.6 The crystal structure of the imidazole adduct of H93G myoglobin has been reported,2 but structural and spectroscopic data for the H93G mutant in the absence of imidazole are not yet available. Interestingly, UV-vis spectra of wild-type metmyoglobin and ferric Coprinus cinereus peroxidase in acidic buffer7,8 bear resemblance to those of the proximal ligand-deficient heme proteins; the native proximal ligation may well be disrupted under such conditions. Among these proximal-ligand mutants, H175G CCP is unique in that its crystal structure indicates two water molecules axial to the heme. However, it is not certain whether one or both of these waters is strongly coordinated, what the water protonation states are, and whether the axial coordination of two water molecules can be maintained in solution. In this study, we have examined the crystal and solution structures of the H175G mutant by UVvis, resonance Raman (RR), and EPR spectroscopy. Ferric H175G CCP9 undergoes a transition from a “red form” near pH 6 to a “green form” near pH 7 (Figure 1). The absorbance at 408 nm (λmax of the red form) changes with pH to give an apparent pKa of ∼6.5 (inset, Figure 1). A fit to these data indicates that the transition involves a cooperative twoproton process. One proton is proposed to be from the ironbound water molecule, but the source of the second proton is unknown. The UV-vis and RR spectra of H175G at pH 10.0 are almost identical to those at pH 7.2 (not shown). FeIII(TMP)s [TMP ) 5,10,15,20-tetrakis(1-methylpyridinium)porphines] exhibit similar ligand ionization in aqueous solution where the five-coordinate iron is ligated by a water at pH 11.10,11 Resonance Raman spectra of heme proteins are dominated by porphyrin skeletal vibrations, i.e., totally symmetric ν2, ν3, and ν4 with Soret excitation, nonsymmetric modes ν10, ν11, and ν19 with Q-band excitation, and ν2 and ν10 with near-UV (∼350 nm) excitation.12-15 The frequencies of these modes are determined by the iron coordination and spin states. The RR spectra of H175G16 are shown in Figure 2. The well-defined ν3 and ν10 frequencies at 1494 and 1630 cm-1, respectively, of the red solution (pH 5.9) are clearly characteristic of a fivecoordinate high-spin (5cHS) ferric heme.15,17-20 The RR spectra of the green solution (pH 7.2) also indicate a dominant 5cHS