Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.     

Chemical nature of the porphyrin pi cation radical in horseradish peroxidase compound I

The electron paramagnetic resonance (EPR) and M?ssbauer properties of native horseradish peroxidase have been compared with those of a synthetic derivative of the enzyme in which a mesohemin residue replaces the natural iron protoporphyrin IX heme prosthetic group. The oxyferryl pi cation radical intermediate, compound I, has been formed from both the native and synthetic enzyme, and the magnetic properties of both intermediates have been examined. The optical absorption characteristics of compound I prepared from mesoheme-substituted horseradish peroxidase are different from those of the compound I prepared from native enzyme [DiNello, R. K., & Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912]. By analogy to model-compound studies, it has been suggested that these optical absorption differences are due to the formation of an A2u and an A1u pi cation radical species, respectively. However, the EPR and M?ssbauer properties of the native and synthetic enzyme and of their oxidized intermediates are quite similar, if not identical, and the data favor an A2u radical for both compounds I.     

Tryptophan-191----phenylalanine, a proximal-side mutation in yeast cytochrome c peroxidase that strongly affects the kinetics of ferrocytochrome c oxidation

On the basis of X-ray structural information, it was previously proposed that tryptophan-191 of yeast cytochrome c peroxidase (CCP) may be important in determining the spectroscopic and catalytic properties of the enzyme [Edwards, S. L., Xuong, Ng. H., Hamlin, R. C., & Kraut, J. (1987) Biochemistry 26, 1503-1511]. By use of site-directed mutagenesis and an Escherichia coli expression system, a mutant phenylalanine-191 (F191) CCP was prepared in order to examine the effects of altering the H-bonding and pi-pi interactions that occur between Trp-191 and the iron-coordinated proximal His-175 in the parent enzyme. The F191 mutant enzyme exhibits a dramatic decrease (approximately 3000-fold at pH 7) in V0/e for catalysis of peroxide-dependent ferrocytochrome c oxidation, while V0/e for oxidation of ferrocyanide is decreased only 4.6-fold compared to that of the parent. The Fe3+/Fe2+ Em,7 and the stability of the oxyferryl center in the H2O2-oxidized mutant enzyme are relatively unaffected by the mutation, but the species responsible for a radical-like signal centered at g = 2.00 has been destabilized approximately 100-fold with respect to spontaneous decay. Steady-state kinetic assays as well as transient-state laser flash photolysis experiments utilizing flavin semiquinones as reductants indicate that the mutant CCP forms a complex with cytochrome c but the oxyferryl center in the oxidized enzyme is no longer able to be rapidly reduced by ferrocytochrome c. The most likely reasons for this kinetic behavior are either that new steric constraints exist in the mutant which impede relaxation of the iron center to the resting ferric state or that the indole ring of Trp-191 is important in a specific interprotein electron-transfer pathway that exists between the heme centers of CCP and cytochrome c.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase

The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.     

Electron transfer between cytochromec and cytochromec peroxidase

The reaction between cytochromec (CC) and cytochromec peroxidase (CcP) is a very attractive system for investigating the fundamental mechanism of biological electron transfer. The resting ferric state of CcP is oxidized by hydrogen peroxide to compound I (CMPI) containing an oxyferryl heme and an indolyl radical cation on Trp-191. CMPI is sequentially reduced to CMPII and then to the resting state CcP by two molecules of CC. In this review we discuss the use of a new ruthenium photoreduction technique and other rapid kinetic techniques to address the following important questions: (1) What is the initial electron acceptor in CMPI? (2) What are the true rates of electron transfer from CC to the radical cation and to the oxyferryl heme? (3) What are the binding domains and pathways for electron transfer from CC to the radical cation and the oxyferryl heme? (4) What is the mechanism for the complete reaction under physiological conditions?     

Rapid-scan stopped-flow studies of the pH dependence of the reaction between mercuric reductase and NADPH

The reaction of NADPH with the flavoenzyme mercuric reductase has been studied by rapid-scan stopped-flow spectrophotometry at 5 degrees C in the pH range 5.1-9.5. An intermediate formed within the dead time of the apparatus, and proposed to be an NADPH complex of oxidized enzyme, has an almost pH-independent spectrum. At pH 5.1 the formation of this species is followed by a rapid bleaching (k = 145 s-1) of the main flavin absorption band at 455 nm concomitantly with an absorbance increase around 395 nm. This process, which has a kinetic hydrogen isotope effect of 2.4, becomes less prominent at higher pH values and is not detectable above pH 7. It is suggested that this process includes the formation of a covalent thiol-flavin C-4a derivative stabilized by protonation of the active site. In the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two-electron-reduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four-electron-reduced enzyme. The spectral changes observed above pH 7 are nearly independent of pH. The first measurable step (k = 48 s-1 at pH 9.5) is thought to include the formation of an NADP+ complex of two-electron-reduced enzyme, while the final step (k = 6.3 s-1 at pH 9.5) results in the above-mentioned NADPH complex with two-electron-reduced enzyme. A minimal kinetic scheme rationalizing the observed pH dependence of the reaction and the observed isotope effects is presented.     

Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity.

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.     

Compound I radical in site-directed mutants of cytochrome c peroxidase as probed by electron paramagnetic resonance and electron-nuclear double resonance.

The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical and transient state kinetic studies on the formation and decomposition of horseradish peroxidase compounds XI and XII

Previous studies on the chlorination reaction catalyzed by horseradish peroxidase using chlorite as the source of chlorine detected the formation of a chlorinating intermediate that was termed Compound X (Shahangian, S., and Hager, L.P. (1982) J. Biol. Chem. 257, 11529-11533). These studies indicated that at pH 10.7, the optical absorption spectrum of Compound X was similar to the spectrum of horseradish peroxidase Compound II. Compound X was shown to be quite stable at alkaline pH values. This study was undertaken to examine the relationship between the oxidation state of the iron protoporphyrin IX heme prosthetic group in Compound X and the chemistry of the halogenating intermediate. The experimental results show that the optical absorption properties and the oxidation state of the heme prosthetic group in horseradish peroxidase are not directly related to the presence of the activated chlorine atom in the intermediate. The oxyferryl porphyrin heme group in alkaline Compound X can be reduced to a ferric heme species that still retains the activated chlorine atom. Furthermore, the reaction of chlorite with horseradish peroxidase at acidic pH leads to the secondary formation of a green intermediate that has the spectral properties of horseradish peroxidase Compound I (Theorell, H. (1941) Enzymologia 10, 250-252). The green intermediate also retains the activated chlorine atom. By analogy to peroxidase Compound I chemistry, the heme prosthetic group in the green chlorinating intermediate must be an oxyferryl porphyrin pi-cation radical species (Roberts, J. E., Hoffman, B. M., Rutter, R. J., and Hager, L. P. (1981) J. Am. Chem. Soc. 103, 7654-7656). To be consistent with traditional peroxidase nomenclature, the red alkaline form of Compound X has been renamed Compound XII, and the green acidic form has been named Compound XI. The transfer of chlorine from the chlorinating intermediate to an acceptor molecule follows an electrophilic (rather than a free radical) path. A mechanism for the reaction is proposed in which the activated chlorine atom is bonded to a heteroatom on an active-site amino acid side chain. Transient state kinetic studies show that the initial intermediate, Compound XII, is formed in a very fast reaction. The second-order rate constant for the formation of Compound XII is approximately 1.1 x 10(7) M-1 s-1. The rate of formation of Compound XII is strongly pH-dependent. At pH 9, the second-order rate constant for the formation of Compound XII drops to 1.5 M-1 s-1. At acidic pH values, Compound XII undergoes a spontaneous first-order decay to yield Compound XI.(ABSTRACT TRUNCATED AT 400 WORDS)     

Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

Using oligonucleotide-directed site-specific mutagenesis, we have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 degrees C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.     

Studies on the equilibria and kinetics of the reactions of ferrous catalase with ligands

The optical absorption spectrum of bovine liver catalase was found to change on light irradiation in the presence of proflavin and EDTA in a deaerated solution. Upon addition of CO to the photolyzed product, the spectrum changed to an another form, suggesting that the photolyzed product is the ferrous form of the enzyme and CO is bound to the ferrous enzyme. When O2 was introduced into the ferrous enzyme, the absorption spectrum returned to its original ferric state. An intermediate spectrum was obtained in this reaction at -20 degrees C in 33% v/v ethylene glycol. Judged from the spectral characteristics of this compound, it is probably an oxyferrous enzyme. It was converted into ferric enzyme gradually when the sample was left at room temperature. The ferrous enzyme, which was generated by flash photolysis of the CO complex of the enzyme in an air-saturated buffer, reacted with O2 to form the oxyferrous enzyme with a second order rate constant of 9.2 x 10(3) M-1.s-1 at pH 8.6 and 20 degrees C. The oxyferrous enzyme thus obtained autodecomposed into the ferric form with a rate constant of 0.1 s-1.     

Role of electrostatics and salt bridges in stabilizing the compound I radical in ascorbate peroxidase

Cytochrome c (CcP) and ascorbate peroxidase (APX) are heme peroxidases which have very similar active site structures yet differ substantially in the properties of compound I, the intermediate formed upon reaction with peroxides. Although both peroxidases have a tryptophan in the proximal heme pocket, Trp191 in CcP and Trp179 in APX, only Trp191 in CcP forms a stable cation radical while APX forms the more traditional porphyrin pi-cation radical. Previous work [Barrows, T. P., et al. (2004)Biochemistry 43, 8826-8834] has shown that converting three methionine residues in the cytochrome c peroxidase (CcP) proximal heme pocket to the corresponding residues in APX dramatically decreased the stability of the Trp191 radical in CcP compound I. On the basis of these results, we reasoned that replacing the analogous residues at positions 160, 203, and 204 in APX with methionine should stabilize a Trp179 radical in APX compound I. Steady- and transient-state kinetics of this mutant (designated APX3M) show a significant destabilization of the native porphyrin pi-radical, while electron paramagnetic resonance (EPR) studies show an increase in the intensity of the signal at g = 2.006 with characteristics consistent with formation of a Trp radical. This hypothesis was tested by replacing Trp179 with Phe in the APX3M background. The EPR spectrum of this mutant was very similar to that of the CcP W191G mutant which is known to form a tyrosine radical. Previously published theoretical studies [Guallar, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6998-7002] suggest that electrostatic shielding of the heme propionates also plays a role in the stability of the porphyrin radical. Arg172 in APX hydrogen bonds with one of the heme propionates. Replacing Arg172 with an asparagine residue in the APX3M background generates a mutant which no longer forms the full complement of the compound I porphyrin pi-radical. These results suggest that the electrostatics of the proximal pocket and the shielding of propionate groups by salt bridges are critical factors controlling the location of a stable compound I radical in heme peroxidases.     

A free radical mechanism of prostaglandin synthase-dependent aminopyrine demethylation

The mechanism of prostaglandin synthase-dependent N-dealkylation has been investigated using an enzyme preparation derived from ram seminal vesicles. Incubation of an N-alkyl substrate, aminopyrine, with enzyme and arachidonic acid, 15-hydroperoxyarachidonic acid, or tert-butyl hydroperoxide resulted in the formation of the transient aminopyrine free radical species. Formation of this radical species, which was detected by electron paramagnetic resonance spectroscopy and/or absorbance at 580 nm, was maximal approximately 30 s following initiation of the reaction and declined thereafter. Free radical formation corresponded closely with formaldehyde formation in this system, in terms of dependence upon substrate and cofactor concentration, as well as in terms of time course. Both aminopyrine free radical and formaldehyde formation were inhibited by indomethacin and flufenamic acid, inhibitors of prostaglandin synthase. The results suggest that the aminopyrine free radical is an intermediate in the prostaglandin synthase-dependent aminopyrine N-demethylase pathway. The aminopyrine free radical electron paramagnetic resonance spectrum revealed that this species is a one-electron oxidized cation radical of the parent compound. A reaction mechanism has been proposed in which aminopyrine undergoes two sequential one-electron oxidations to an iminium cation, which is then hydrolyzed to the demethylated amine and formaldehyde. Accordingly, the oxygen atom of the aldehyde product is derived from neither molecular nor hydroperoxide oxygen, but from water.     

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.     

Evidence for radical formation at Tyr-353 in Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis KatG is a heme-containing catalase-peroxidase responsible for activation, through its peroxidase cycle, of the front line antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide). Formation of Compound I (oxyferryl heme-porphyrin pi-cation radical), the classical peroxidase intermediate generated when the resting enzyme turns over with alkyl peroxides, is rapidly followed by production of a protein-centered tyrosyl radical in this enzyme. In our efforts to identify the residue at which this radical is formed, nitric oxide was used as a radical scavenging reagent. Quenching of the tyrosyl radical generated in the presence of NO was shown using electron paramagnetic resonance spectroscopy, and formation of nitrotyrosine was confirmed by proteolytic digestion followed by high performance liquid chromatography analysis of the NO-treated enzyme. These results are consistent with formation of nitrosyltyrosine by addition of NO to tyrosyl radical and oxidation of this intermediate to nitrotyrosine. Two predominant nitrotyrosine-containing peptides were identified that were purified and sequenced by Edman degradation. Both peptides were derived from the same M. tuberculosis KatG sequence spanning residues 346-356 with the amino acid sequence SPAGAWQYTAK, and both peptides contained nitrotyrosine at residue 353. Some modification of Trp-351 most probably into nitrosotryptophan was also found in one of the two peptides. Control experiments using denatured KatG or carried out in the absence of peroxide did not produce nitrotyrosine. In the mutant enzyme KatG(Y353F), which was constructed using site-directed mutagenesis, a tyrosyl radical was also formed upon turnover with peroxide but in poor yield compared with wild-type KatG. Residue Tyr-353 is unique to M. tuberculosis KatG and may play a special role in the function of this enzyme.     

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.     

Electrostatic control of the tryptophan radical in cytochrome c peroxidase

Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.     

Rapid-scan stopped-flow studies of the flavoenzyme mercuric reductase during catalytic turnover

Time-resolved absorption spectra of the FAD-containing enzyme mercuric reductase were recorded during the catalytic reaction at 25 degrees C, pH 7.3. With an excess of NADPH over Hg2+ there was a rapid (k = 43 s-1) initial formation of a spectral species similar to that previously assigned to an NADPH complex of two-electron-reduced enzyme, EH2-NADPH. This spectrum persisted during the quasisteady-state phase of the reaction suggesting that EH2-NADPH is a true catalytic intermediate and that the rate of catalysis is limited by the oxidation of EH2-NADPH by Hg2+. Also with an excess of Hg2+ over NADPH a spectrum similar to that of EH2-NADPH was rapidly formed. As the NADPH was exhausted, the spectrum of oxidized enzyme, E, did not reappear but rather a spectrum similar to that previously assigned to an NADP+ complex of two-electron-reduced enzyme, EH2-NADP+. These results suggest that EH2-HADP+ cannot rapidly reduce the Hg2+ substrate. However, eventually all reducing equivalents from NADPH added to oxidized, activated enzyme are utilized for the reduction of Hg2+. A mechanism model is proposed that does not involve the free, oxidized enzyme in the catalytic cycle.