Impact of Proximal and Distal Pocket Site-Directed Mutations on the Ferric/Ferrous Heme Redox Potential of Yeast Cytochrome-c-Peroxidase.

Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~-180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, and measurements made in a bis-tris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52, and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential are also explored as a pathway for model building.

A distal histidine mutant (H52Q) of yeast cytochrome c peroxidase catalyzes the oxidation of H(2)O(2) instead of its reduction.

A H52Q variant of yeast cytochrome c peroxidase (CcP), in which the distal histidine is replaced by glutamine, catalyzes oxidation of H(2)O(2) instead of reduction. This redirection of catalytic action is detected by protein film voltammetry. In the presence of H(2)O(2), wild-type CcP, adsorbed on a graphite electrode, shows a strong catalytic reduction wave commencing at about 0.8V (pH 5.4); by contrast, H52Q does not exhibit this activity but instead shows a catalytic oxidation current at potentials in the region of 0.9 V. The oxidation current is partly suppressed in the presence of tetranitromethane (a superoxide scavenger) and is not observed for other mutants studied, including H52A. The only significant structural change in the H52Q variant is that the Q-52 side chain occupies the space vacated by the H-52 imidazole; specifically, the N-epsilon atom that is believed to transfer a proton and induce O--O cleavage is replaced, to within 0.75 A, by the carbamide-O. Thus, while the weakly basic amide functionality is unable to serve in the reorganization of bound H(2)O(2), it is able to facilitate its oxidation, most obviously by serving as a H-bond acceptor to assist formation of a labile superoxide intermediate.

Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 1. Effects on structure.

Replacement of the axial histidine ligand with exogenous imidazole has been accomplished in a number of heme protein mutants, where it often serves to complement the functional properties of the protein. In this paper, we describe the effects of pH and buffer ion on the crystal structure of the H175G mutant of cytochrome c peroxidase, in which the histidine tether between the heme and the protein backbone is replaced by bound imidazole. The structures show that imidazole can occupy the proximal H175G cavity under a number of experimental conditions, but that the details of the interaction with the protein and the coordination to the heme are markedly dependent on conditions. Replacement of the tethered histidine ligand with imidazole permits the heme to shift slightly in its pocket, allowing it to adopt either a planar or distally domed conformation. H175G crystallized from both high phosphate and imidazole concentrations exists as a novel, 5-coordinate phosphate bound state, in which the proximal imidazole is dissociated and the distal phosphate is coordinated to the iron. To accommodate this bound phosphate, the side chains of His-52 and Asn-82 alter their positions and a significant conformational change in the surrounding protein backbone occurs. In the absence of phosphate, imidazole binds to the proximal H175G cavity in a pH-dependent fashion. At pH 7, imidazole is directly coordinated to the heme (d(Fe--Im) = 2.0 A) with a nearby distal water (d(Fe--HOH) = 2.4 A). This is similar to the structure of WT CCP except that the iron lies closer in the heme plane, and the hydrogen bond between imidazole and Asp-235 (d(Im--Asp) = 3.1 A) is longer than for WT CCP (d(His--Asp) = 2.9 A). As the pH is dropped to 5, imidazole dissociates from the heme (d(Fe--Im) = 2.9 A), but remains in the proximal cavity where it is strongly hydrogen bonded to Asp-235 (d(Im--Asp) = 2.8 A). In addition, the heme is significantly domed toward the distal pocket where it may coordinate a water molecule. Finally, the structure of H175G/Im, pH 6, at low temperature (100 K) is very similar to that at room temperature, except that the water above the distal heme face is not present. This study concludes that steric restrictions imposed by the covalently tethered histidine restrain the heme and its ligand coordination from distortions that would arise in the absence of the restricted tether. Coupled with the functional and spectroscopic properties described in the following paper in this issue, these structures help to illustrate how the delicate and critical interactions between protein, ligand, and metal modulate the function of heme enzymes.

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function.

The inability of imidazole to complement function in the axial histidine deletion mutant, H175G, of yeast cytochrome c peroxidase has been an intriguing but unresolved issue that impacts our understanding of the role of axial ligands in heme catalysis. Here we report the functional and spectroscopic properties of H175G and of its complexes with imidazole. Combined with the crystal structures for these complexes, the data provide a detailed and consistent account of the modes of Im binding in the H175G cavity and their dependence on buffer and pH. UV--vis, EPR, and resonance Raman spectra reveal multiple coordination states for H175G/Im which can be correlated with the crystal structures to assign the following heme environments: H175G/H(2)O/H(2)O, H175G/Im(d)/phosphate(c), H175G/Im(d)/H(2)O(c), H175G/Im(c)/H(2)O(d), and H175G/Im(c)/OH(-)(c), where H175G/X/Y defines the proximal species as X and the distal species as Y and c and d subscripts refer, where known, to the coordinated and dissociated states, respectively. Resonance Raman data for reduced H175G/Im show two substates for heme-coordinated Im differing in the strength of their hydrogen bond to Asp-235, in a fashion similar to WT CCP. NO binding to ferrous H175G/Im results in dissociation of Im from the heme but not from the cavity, while no dissociation is observed for WT CCP, indicating that steric tethering may, in part, control NO-induced dissociation of trans ligands. H175G/Im forms an oxidized compound I state with two distinct radical species, each with a dramatically different anisotropy and spin relaxation from that of the Trp-191 radical of WT CCP. It is suggested that these signals arise from alternate conformations of Trp191 having different degrees of exchange coupling to the ferryl heme, possibly mediated by the conformational heterogeneity of Im within the H175G cavity. The kinetics of the reaction of H175G/Im with H(2)O(2) are multiphasic, also reflecting the multiple coordination states of Im. The rate of the fastest phase is essentially identical to that of WT CCP, indicating that the H175G/Im(c)/H(2)O(d) state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is <5% of WT and is unaffected by Im coordination. In summary, Im coordination to H175G results in a number of conformers, one of which is structurally and spectroscopically very similar to WT CCP. However, while this form is fully reactive with peroxide, the reaction with cytochrome c remains inefficient, perhaps implicating the altered Trp-191 radical species.

Investigating resins for solid phase organic synthesis: the relationship between swelling and microenvironment as probed by EPR and fluorescence spectroscopy.

The relationship between observed swelling of two cross-linked polystyrene resins and the microenvironment within polymer matrixes has been examined. Polystyrene cross-linked with either divinyl benzene (Merrifield resin) or 1,4-bis(4-vinylphenoxy)butane (JandaJel) was investigated with fluorescence and electron-paramagnetic resonance spectroscopy. Fluorescence spectroscopy revealed a superior correlation between observed swelling and solvation effects using a dansyl probe with JandaJel than with Merrifield resin. However, the internal viscosity of pre-swollen JandaJel is higher than Merrifield resin, as determined by EPR measurements. The combination of these two analytical methods provides insights into the physical differences observed between these two chemically similar resins and suggests caution should be used if using singular physical techniques to probe the microenvironment of polymeric matrixes.

Unusual oxidative chemistry of N(omega)-hydroxyarginine and N-hydroxyguanidine catalyzed at an engineered cavity in a heme peroxidase.

Heme enzymes are capable of catalyzing a range of oxidative chemistry with high specificity, depending on the surrounding protein environment. We describe here a reaction catalyzed by a mutant of cytochrome c peroxidase, which is similar but distinct from those catalyzed by nitric-oxide synthase. In the R48A mutant, an expanded water-filled cavity was created above the distal heme face. N-hydroxyguanidine (NHG) but not guanidine was shown to bind in the cavity with K(d) = 8.5 mM, and coordinate to the heme to give a low spin state. Reaction of R48A with peroxide produced a Fe(IV)=O/Trp(.+) center capable of oxidizing either NHG or N(omega)-hydroxyarginine (NHA), but not arginine or guanidine, by a multi-turnover catalytic process. Oxidation of either NHG or NHA by R48A did not result in the accumulation of NO, NO(2)(-), NO(3)(-), urea, or citrulline, but instead afforded a yellow product with absorption maxima of 257 and 400 nm. Mass spectrometry of the derivatized NHA products identified the yellow species as N-nitrosoarginine. We suggest that a nitrosylating agent, possibly derived from HNO, is produced by the oxidation of one molecule of substrate. This then reacts with a second substrate molecule to form the observed N-nitroso products. This complex chemistry illustrates how the active sites of enzymes such as nitric-oxide synthase may serve to prevent alternative reactions from occurring, in addition to enabling those desired.

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH.

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4 degrees C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP (MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met-Lys-Thr (MKT) at the N-terminus, CCP (MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP (MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP (MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP (MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (< or = 5%) in the spectrum of CCP (MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (lambda CT = 637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP (MKT) (lambda CT = 637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.

Influence of protein environment on magnetic circular dichroism spectral properties of ferric and ferrous ligand complexes of yeast cytochrome c peroxidase.

The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4 degrees C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b5, a known bis-histidine complex. Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP.

Oxidized and reduced Azotobacter vinelandii ferredoxin I at 1.4 A resolution: conformational change of surface residues without significant change in the [3Fe-4S]+/0 cluster.

The refined structure of reduced Azotobacter vinelandii 7Fe ferredoxin FdI at 100 K and 1.4 A resolution is reported, permitting comparison of [3Fe-4S]+ and [3Fe-4S]0 clusters in the same protein at near atomic resolution. The reduced state of the [3Fe-4S]0 cluster is established by single-crystal EPR following data collection. Redundant structures are refined to establish the reproducibility and accuracy of the results for both oxidation states. The structure of the [4Fe-4S]2+ cluster in four independently determined FdI structures is the same within the range of derived standard uncertainties, providing an internal control on the experimental methods and the refinement results. The structures of the [3Fe-4S]+ and [3Fe-4S]0 clusters are also the same within experimental error, indicating that the protein may be enforcing an entatic state upon this cluster, facilitating electron-transfer reactions. The structure of the FdI [3Fe-4S]0 cluster allows direct comparison with the structure of a well-characterized [Fe3S4]0 synthetic analogue compound. The [3Fe-4S]0 cluster displays significant distortions with respect to the [Fe3S4]0 analogue, further suggesting that the observed [3Fe-4S]+/0 geometry in FdI may represent an entatic state. Comparison of oxidized and reduced FdI reveals conformational changes at the protein surface in response to reduction of the [3Fe-4S]+/0 cluster. The carboxyl group of Asp15 rotates approximately 90 degrees, Lys84, a residue hydrogen bonded to Asp15, adopts a single conformation, and additional H2O molecules become ordered. These structural changes imply a mechanism for H+ transfer to the [3Fe-4S]0 cluster in agreement with electrochemical and spectroscopic results.

Copper binding to the prion protein: structural implications of four identical cooperative binding sites.

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd approximately 6 microM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nepsilon2 and Ndelta1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.

Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase.

The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.

Protein conformer selection by ligand binding observed with crystallography.

A large-scale movement between "closed" and "open" conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of Trp191 in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop Pro190-Asn195 and exposing Trp191 to the protein surface. Kinetic measurements are consistent with a two-step binding mechanism in which the rate-limiting step is a transition of the protein to the open state, which then binds the ligand. This large-scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild-type protein is in the open form in solution at any given time.

Introduction of novel substrate oxidation into cytochrome c peroxidase by cavity complementation: oxidation of 2-aminothiazole and covalent modification of the enzyme.

The binding and oxidation of an artificial substrate, 2-aminothiazole, by an engineered cavity of cytochrome c peroxidase is described. The W191G mutant has been shown to create a buried cavity into which a number of small heterocyclic compounds will bind [Fitzgerald, M. M., Churchill, M. J., McRee, D. E., & Goodin, D. B. (1994) Biochemistry 33, 3807-3818], providing a specific site near the heme from which substrates might be oxidized. In this study, we show by titration calorimetry that 2-aminothiazole binds to W191G with a Kd of 0.028 mM at pH 6. A crystal structure at 2.3 A resolution of W191G in the presence of 2-aminothiazole reveals the occupation of this compound in the cavity, and indicates that it is in van der Waals contact with the heme. The WT enzyme reacts with H2O2 to form Compound ES, in which both the iron center and the Trp-191 side chain are reversibly oxidized. For the W191F (and perhaps the W191G) mutants, the iron is still oxidized, but the second equivalent exists transiently as a radical on the porphyrin before migrating to an alternate protein radical site [Erman, J. E., Vitello, L. B., Mauro, J. M., & Kraut, J. (1989) Biochemistry 28, 7992-7995]. Two separate reactions are observed between 2-aminothiazole and the oxidized centers of W191G. In the one reaction, optical and EPR spectra of the heme are used to show that 2-aminothiazole acts as an electron donor to the ferryl (Fe4+&dbd;O) center of W191G to reduce it to the ferric oxidation state. This reaction occurs from within the cavity, as it is not observed for variants that lack this artificial binding site. A second reaction between 2-aminothiazole and peroxide-oxidized W191G, which is much less efficient, results in the specific covalent modification of Tyr-236. Electrospray mass spectra of the W191G after incubation in 2-aminothiazole and H2O2 show a modification of the protein indicative of covalent binding of 2-aminothiazole. The site of modification was determined to be Tyr-236 by CNBr peptide mapping and automated peptide sequencing. The covalent modification is only observed for W191G and W191F which form the alternate radical center. This observation provides an unanticipated assignment of this free radical species to Tyr-236, which is consistent with previous proposals that it is a tyrosine. The oxidation of 2-aminothiazole by W191G represents an example of how the oxidative capacity inherent in the heme prosthetic group and the specific binding behavior of artificial protein cavities can be harnessed and redirected toward the oxidation of organic substrates.

Effect of axial ligand plane reorientation on electronic and electrochemical properties observed in the A67V mutant of rat cytochrome b5.

Mutational studies directed at evaluating the effect of the axial ligand plane orientation on electrochemical properties of cytochrome b5 have been performed. As described in the previous paper, structural consequences of one of these mutations, the A67V mutation, have been evaluated using NMR solution methods. The lack of large shifts relative to the wild-type protein in both the imidazole Ndelta nitrogen and proton resonances of the H63 imidazole ring indicates that the hydrogen bond between the carbonyl of F58 and the imidazole ring of H63 remains intact in this mutant. Effects of the imidazole plane reorientation on the Fe d-orbitals were evaluated on the basis of interpretation of EPR spectra, near-infrared bands associated with ligand-to-metal charge transfer transitions, reorientation of the anisotropy of the paramagnetic center determined by calculation of pseudocontact shifts, and the temperature dependence of the contact-shifted resonances. The dominant effect of the imidazole reorientation appears to have been a destabilization of the d(xz) orbital energy and a reorientation of the d(pi) orbitals. This is surprising in light of the -20 mV shift in the reduction potential of the mutant relative to the wild-type protein and indicates that a destabilization of d(yz)-orbital energy level of the reduced state dictates the observed change in reduction potential. Measured values for the reorganizational energy and heterogeneous electron transfer rates were indistinguishable for wild-type and mutant proteins. This is perhaps surprising, given significant differences in the pattern of electron delocalization into the porphyrin ring observed as significantly altered contact shift patterns. Mutational studies perturbing the H39 imidazole were also performed but with more limited success.

A ligand-gated, hinged loop rearrangement opens a channel to a buried artificial protein cavity.

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.

Altering substrate specificity at the heme edge of cytochrome c peroxidase.

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.

The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochrome c peroxidase.

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.

Horseradish peroxidase Phe172-->Tyr mutant. Sequential formation of compound I with a porphyrin radical cation and a protein radical.

A gene coding for the F172Y mutant of horseradish peroxidase isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperda (SF-9) and Trichoplusia ni egg cell homogenate (HighFive) cells. Homology modeling with respect to three peroxidases for which crystal structures are available places Phe172 on the proximal side of the heme in the vicinity of porphyrin pyrrole ring C. The pH optimum and spectroscopic properties of the F172Y mutant are essentially identical to those of wild type HRP. Vmax values show that the mutant protein retains most of the guaiacol oxidizing activity. Stopped flow studies indicate that Compound I is formed with H2O2 at the same rate (kappa 1 = 1.6 x 10(7) M-1 s-1) at both pH 6.0 and 8.0 as it is with the wild type enzyme. This Compound I species decays rapidly at a rate kappa 2 = 1.01 s-1, pH 7.0, to a second two-electron oxidized species that retains the ferryl (FeIV = O) absorption. EPR studies establish that a ferryl porphyrin radical cation is present in the initial Compound I, but electron transfer from the protein results in formation of a second Compound I species with an unpaired electron on the protein (presumably on Tyr172). The presence or absence of oxidizable amino acids adjacent to the heme is thus a key determinant of whether the second oxidation equivalent in Compound I is found as a porphyrin or protein radical cation.

Identification of a porphyrin pi cation radical in ascorbate peroxidase compound I.

Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the ascorbate peroxidase Fe3+ resting state and to compare the reaction product between the enzyme and H2O2, compound I, with that of cytochrome c peroxidase. Because ascorbate peroxidase has a Trp residue in the proximal heme pocket at the same location as the Trp191 compound I free radical in cytochrome c peroxidase [Patterson, W. R., & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], it was anticipated that ascorbate peroxidase compound I might also contain a Trp-centered radical. However, the ascorbate peroxidase compound I EPR spectrum is totally different from that of cytochrome c peroxidase. Immediately after the addition of H2O2, the 7.5 K EPR spectrum of ascorbate peroxidase compound I exhibits an axial resonance extending from g perpendicular = 3.27 to g parallel approximately 2 that disappears within 30 s, presumably due to endogenous reduction of compound I. In contrast, cytochrome c peroxidase compound I exhibits a long-lived g approximately 2 signal associated with the Trp191 cation free-radical [Houseman, A. L. P., et al. (1993) Biochemistry 32, 4430-4443]. Recently, the 2 K EPR spectrum of a catalase compound I was found to exhibit a broad signal extending from g perpendicular = 3.45 to g parallel approximately 2 and was interpreted as a porphyrin pi cation radical [Benecky, M. J., et al. (1993) Biochemistry 32, 11929-11933]. On the basis of these comparisons, we conclude that ascorbate peroxidase forms an unstable compound I porphyrin pi cation radical, even though it has a Trp residue positioned precisely where the Trp191 radical is located in cytochrome c peroxidase.