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)     

Crystal structure of nitric oxide inhibited cytochrome c peroxidase

We have collected X-ray diffraction data from a crystal of cytochrome c peroxidase (CCP) complexed with the inhibitor nitric oxide to a resolution of 2.55 A. A difference Fourier map shows density indicating the NO ligand is bound to the heme iron at the sixth coordination site in a bent configuration. Structural adjustments were determined by least-squares refinement that yielded an agreement residual of R = 0.18. The orientation of the ligand, tilting toward Arg-48, causes adjustment in the position of this nearby polar side chain. As a model for the substrate hydrogen peroxide, this geometry is consistent with the suggestion that Arg-48 serves to polarize the O-O peroxide bond to promote heterolytic cleavage of the bond [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. Strong difference density is also observed near residues 190-194, especially around the indole ring of Trp-191. The density indicates movement of the indole ring away from the proximal His-175 imidazole ring by about 0.25 A, which appears to cause perturbation of the neighboring residues. The response of Trp-191 on the proximal side of the heme to binding nitric oxide on the distal side probably results from delocalization of the electron density of the ligand. Relevant to this is the recent finding that a mutant in which Trp-191 is replaced by phenylalanine has dramatically reduced activity, less than 0.05% of the parent activity [Mauro, J. M., Fishel, L. A., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256].(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of yeast cytochrome c peroxidase refined at 1.7-A resolution

The crystal structure of cytochrome c peroxidase (EC 1.11.1.5) has been refined to an R factor of 0.20 computed for all reflections to 1.7 A. The refined molecular model includes 263 bound water molecules and allows for x-ray scattering by amorphous solvent. The mean positional error in atomic coordinates is estimated to lie between 0.12 and 0.21 A. Two factors are identified which may account for the ability of the enzyme to stabilize high-oxidation states of the heme iron during catalysis: 1) the proximal histidine forms a hydrogen bond with a buried aspartic acid side chain, Asp-235; and 2) the heme environment is more polar than in the cytochromes c or globins, owing to the presence of the partially buried side-chain of Arg-48 and five water molecules bound in close proximity to the heme. Two of these occupy the presumed peroxide-binding site. Two candidates are likely for the side chain that is oxidized to a free radical during formation of Compound I: 1) Trp-51, which rests 3.3 A above the heme plane in close proximity (2.7 A) to the sixth coordination position; and 2) Met-172, which is 3.7 A from the heme. Nucleophilic stabilization of the methionyl cation radical may be possible via Asp-235. His-181 is found to lie coplanar with the heme in a niche between the two propionates near the suspected cytochrome c-binding site. A network of hydrogen bonds involving this histidine may provide a preferred pathway for electron transfer between hemes.     

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.     

X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli [Fishel, L.A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360], has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53----Ile substitution in CCP(MI). A Trp-51----Phe mutant remains pentacoordinated and exhibits only minor distal structural adjustments. The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron [Smulevich, G., Mauro, J.M., Fishel, L. A., English, A. M., Kraut, J., & Spiro, T. G. (1988) Biochemistry 27, 5477-5485]. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191----Phe mutant that has substantially diminished enzyme activity and altered magnetic properties [Mauro, J. M., Fishel, L. F., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256] accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large (ca. 1 A) local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structure rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. Changing proximal Asp-235 to Asn results in two significant localized structural changes. First, the heme iron moves toward the porphyrin plane, and distal water 595 now clearly resides in the iron coordination sphere at a distance of 2.0 A. The observation of hexacoordinated iron for the D235N mutant is in accord with previous resonance Raman results. Second, the indole side chain of Trp-191 has flipped over as a result of the mutation; the tryptophan N epsilon takes part in a new hydrogen bond with the backbone carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ligand binding and structural perturbations in cytochrome c peroxidase. A crystallographic study

Crystal structures of the complexes formed between cytochrome c peroxidase and cyanide, nitric oxide, carbon monoxide, and fluoride have been determined and refined to 1.85 A. In all four complexes significant changes occur in the distal heme pocket due to movement of Arg-48, His-52, and a rearrangement of active site water molecules. In the cyanide, nitric oxide, and carbon monoxide complexes, Arg-48 moves away from the ligand while in the fluoride complex Arg-48 moves in toward the ligand to form a hydrogen bond or ion pair with the fluoride. More subtle changes occur on the proximal side of the heme. In an earlier study at lower resolution (Edwards, S. L., Kraut, J., and Poulos, T. L. (1988) Biochemistry 27, 8074-8081), we found that nitric oxide binding causes perturbations in the proximal domain involving Trp-191 which has been confirmed by the present study. Trp-191 is stacked parallel to and in contact with the proximal ligand, His-175. Nitric oxide binding results in a slight movement of Trp-191 away from His-175 and a large increase in crystallographic temperature factors indicating increased mobility of these residues on the proximal side of the heme. These proximal-side changes are unique to nitric oxide and are not related strictly to spin-state or oxidation state of the iron atom since similar changes were not observed in the cyanide (low-spin ferric), carbon monoxide (low-spin ferrous), or fluoride (high-spin ferric) complexes.     

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.     

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.     

Kinetic resolution of a tryptophan-radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.     

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)     

Chloroperoxidase compound I: Electron paramagnetic resonance and M?ssbauer studies

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. M?ssbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and M?ssbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and M?ssbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.     

Detection of an oxyferryl porphyrin pi-cation-radical intermediate in the reaction between hydrogen peroxide and a mutant yeast cytochrome c peroxidase. Evidence for tryptophan-191 involvement in the radical site of compound I

Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of the native yeast enzyme. This spectrum is characteristic of an oxyferryl, Fe(IV), heme. Stopped-flow studies reveal that the reaction between the mutant enzyme and hydrogen peroxide is biphasic with the transient formation of an intermediate whose absorption spectrum is quite distinct from that of either the native ferric enzyme or the final product. Rapid spectral scanning of the intermediate provides a spectrum characteristic of an oxyferryl porphyrin pi-cation-radical species. At pH 6, 100 mM ionic strength, and 25 degrees C, the rate constant for formation of the oxyferryl pi-cation radical has a lower limit of 6 X 10(7) M-1 s-1 and the rate of conversion of the transient intermediate to the final oxidized product is 51 +/- 4 s-1. Evidence is presented indicating that Trp-191 either is the site of the radical in CcP compound I or is intimately involved in formation of the radical.     

Specific Function of the Met-Tyr-Trp Adduct Radical and Residues Arg-418 and Asp-137 in the Atypical Catalase Reaction of Catalase-Peroxidase KatG

Catalase activity of the dual-function heme enzyme catalase-peroxidase (KatG) depends on several structural elements, including a unique adduct formed from covalently linked side chains of three conserved amino acids (Met-255, Tyr-229, and Trp-107, Mycobacterium tuberculosis KatG numbering) (MYW). Mutagenesis, electron paramagnetic resonance, and optical stopped-flow experiments, along with calculations using density functional theory (DFT) methods revealed the basis of the requirement for a radical on the MYW-adduct, for oxyferrous heme, and for conserved residues Arg-418 and Asp-137 in the rapid catalase reaction. The participation of an oxyferrous heme intermediate (dioxyheme) throughout the pH range of catalase activity is suggested from our finding that carbon monoxide inhibits the activity at both acidic and alkaline pH. In the presence of H₂O₂, the MYW-adduct radical is formed normally in KatG[D137S] but this mutant is defective in forming dioxyheme and lacks catalase activity. KatG[R418L] is also catalase deficient but exhibits normal formation of the adduct radical and dioxyheme. Both mutants exhibit a coincidence between MYW-adduct radical persistence and H₂O₂ consumption as a function of time, and enhanced subunit oligomerization during turnover, suggesting that the two mutations disrupting catalase turnover allow increased migration of the MYW-adduct radical to protein surface residues. DFT calculations showed that an interaction between the side chain of residue Arg-418 and Tyr-229 in the MYW-adduct radical favors reaction of the radical with the adjacent dioxyheme intermediate present throughout turnover in WT KatG. Release of molecular oxygen and regeneration of resting enzyme are thereby catalyzed in the last step of a proposed catalase reaction.     

Raman spectroscopic characterization of tryptophan side chains in lysozyme bound to inhibitors: role of the hydrophobic box in the enzymatic function.

The state of H-bonding and the hydrophobic interaction of six tryptophan side chains in lysozyme bound to substrate-analogous inhibitors were investigated by combining H----D exchange labeling and Raman difference spectroscopy. The frequency of the W17 band due to Trp-63 shifts downward upon inhibitor binding, indicating a specific and strong H-bond formation between the N1 site of the side chain and the inhibitor molecule. On the other hand, the H-bonding state of Trp-62 in the complex is as weak as that in inhibitor-free lysozyme, suggesting no contribution of this residue to the inhibitor binding. Intensity increases of W17 and W18 bands observed upon inhibitor binding are, respectively, ascribed to an increase at Trp-28 and a decrease at Trp-111 in hydrophobic interactions with the environment. The environmental changes are explained consistently by a movement of the Met-105 side chain sandwiched by two indole rings of Trp-28 and 111 in the direction from Trp-111 to Trp-28. The sandwich structure in a core domain, hydrophobic box, and its rearrangement are considered to play an important role in the enzymatic function of lysozyme.     

Conformational and functional studies of chemically modified cytochromes: N-bromosuccinimide- and formyl-cytochromes c.

N-bromosuccinimide-cytochromes c (Myer, Y. P. (1972), Biochemistry 11, 4195) and formyl-cytochrome c (Aviram, I and Schejter, A. (1971), Biochim. Biophys. Acta 229, 113) have been chromatographically purified, and the resulting components have been characterized in terms of their structure, conformation, and function. The activity measurements are considered in terms of the oxidizability, as the transference of an electron to solubilized cytochrome c oxidase, and reducibility, as the tendency to accept an electron from NADH-cytochrome c reductase. Conformational characterization has been carried out by absorption measurements, pH-spectroscopic behavior, circular dichroism, thermal denaturation, ionization of phenolic hydroxyls, the tendency to form the CO complex, and autoxidation with molecular oxygen. NBS-cytochrome c yields two major components, the relative proportions of which, with increasing modification of the protein, exhibit a pattern typical of the formation of the two in a consecutive manner. The first product contains the modification of the Trp-59 and Met-65 side chains, and the second contains the added modification of Met-80. The former in both valence states of iron is more or less like the native protein, except for an apparently slightly loosened heme crevice; the latter, as in other modifications involving modification of centrally coordinated Met-80, was found to be in a conformational state characteristic of the native protein with a disrupted central coordination complex, a loosened heme crevice, and small, but finite derangement of the polypeptide conformation. Functionally, the first component reflected 55% of the reducibility property and an unimpaired oxidizability property, while the latter exhibited derangement of both aspects of cytochrome c activity. Formyl-cytochrome c yielded a single component with modification of Trp-59. Conformationally, in both valence states, it is a molecular form with a disrupted central coordination complex, a loosened heme crevice, and gross derangement of the overall protein conformation. It exhibits a minimal reducibility property, 12%, whereas it retains a native-like tendency to transfer an electron to cytochrome c oxidase. The data from the NBS-cytochrome c components are analyzed with reference to the two forms in the earlier studies of the unpurified preparations. The results are found to be in agreement with one another. The selectivity between the reducibility and the oxidizability exhibited by the first NBS component and formyl-cytochrome c, irrespective of significant differences in the conformational and coordinational configurations of the two, has been viewed in light of a two-path, two-function model for oxidoreduction, as well as with reference to conformational and structural requirements for the oxidizability and reducibility properties of the molecule.     

Regulation of the properties of the heme-NO complexes in nitric-oxide synthase by hydrogen bonding to the proximal cysteine

Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline from l-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal isoform of NOS (nNOS)) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the Trp-409 mutants as compared with the wild type enzyme, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the Trp-409 mutants (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907-26911). The optical and resonance Raman spectra of the Fe2+NO complexes of the Trp-409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate Fe2+NO complex is present. These data show that the hydrogen bond provided by the Trp-409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heme. Taken together our results indicate that the heme environment on the proximal side of nNOS is critical for the formation of a stable iron-cysteine bond and for the control of the electronic properties of heme-NO complexes.     

Assignment of individual heme EPR signals of Desulfovibrio baculatus (strain 9974) tetraheme cytochrome c3. A redox equilibria study

An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmx = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results.     

Myeloperoxidase of the leukocyte of normal blood. Nature of the prosthetic group of myeloperoxidase

The absorption spectra of alkaline pyridine hemochrome of myeloperoxidase in its native, acid, and modified forms were similar to those of heme a, and the molar extinction coefficient of myeloperoxidase heme was very similar to that of heme a, assuming that myeloperoxidase contains only one heme. The anaerobic titration of myeloperoxidase with dithionite showed that one electron was consumed per molecule of the enzyme for its conversion to its reduced form. The EPR spectrum of myeloperoxidase indicated that the enzyme contains both high-spin heme and non-heme iron. Carbonyl reagents, such as borohydride, hydrazine, and benzhydrazide, reacted with myeloperoxidase, causing blue shifts in its absorption spectrum. The heme was labeled with a tritium of boro[3H]hydride, suggesting that the reagents reacted with a formyl group on the porphyrin ring of the myeloperoxidase heme. When hydrazine was added to cyanide complex I of myeloperoxidase the complex was converted to the hydrazine-enzyme compound. Myeloperoxidase reacted with bisulfite to form a compound with an absorption spectrum similar to that of cyanide complex I. Borohydride-treated myeloperoxidase formed only one cyanide complex, while the native enzyme formed two different cyanide complexes, I (Kd = 0.3 muM) and II (approximate Kd = 0.1 mM). The EPR spectrum indicated that cyanide complex I of myeloperoxidase still contained high-spin heme. The results suggested that cyanide complex I and the bisulfite compound of myeloperoxidase were adducts between the nucleophilic reagents and the formyl group of myeloperoxidase heme. Based on these results, we concluded that one of the two iron atoms in a myeloperoxidase molecule exists in a formyl-heme moiety similar to heme a and the other exists as a non-heme iron.     

Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione,NADH, ascorbate,and dichlorofluorescin by cytochrome c/H2O2. Implications for oxidative stress during apoptosis

The release of cytochrome c from mitochondria is a crucial step in apoptosis, resulting in the activation of the caspase proteases. A further consequence of cytochrome c release is the enhanced mitochondrial production of superoxide radicals (O2.), which are converted to hydrogen peroxide by manganese-superoxide dismutase. Recently, we showed that cytochrome c is a potent catalyst of 2',7'-dichlorofluorescin oxidation to the fluorescent 2',7'-dichlorofluorescein by these species, leading to the conclusion that 2',7'-dichlorofluorescein fluorescence is a reflection of cytosolic cytochrome c concentration rather than "reactive oxygen species" levels (Burkitt, M. J., and Wardman, P. (2001) Biochem. Biophys. Res. Commun. 282, 329-333). The oxidant generated from cytochrome c has so far not been identified. Several authors have suggested that the hydroxyl radical (*OH) is generated, but others have discussed the possibility of a peroxidase compound I. By examining the effects of various antioxidants (glutathione, ascorbate, and NADH) and "hydroxyl radical scavengers" (ethanol and mannitol) on the rate of 2',7'-dichlorofluorescin oxidation by cytochrome c, together with complementary EPR spin-trapping studies, we demonstrate that the hydroxyl radical is not generated. Instead, our findings suggest the formation of a peroxidase compound I-type intermediate, in which one oxidizing equivalent is present as an oxoferryl heme species and the other as the protein tyrosyl radical previously identified (Barr, D. P., Gunther, M. R., Deterding, L. J., Tomer, K. B., and Mason, R. P. (1996) J. Biol. Chem. 271, 15498-15503). Competition studies involving spin traps indicated that the oxoferryl heme component is the active oxidant. These findings provide an improved understanding of the physicochemical basis of the redox changes that occur during apoptosis.