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.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

‘Lipidic particle’ systems as visualized by thin-section electron microscopy

Lipidic phases, containing ‘lipidic particles’ (cardiolipin/egg lecithin in the presence of Ca²⁺ and egg lecithin/dioleoylphosphatidylcholine/cholesterol) have been investigated with an alternative thin-section method, using rapid cryofixation and freeze substitution according to Müller et al. (Müller, M., Marti, T. and Kriz, S. (1980) in Proc. 7th Eur. Congr. on Electron Microscopy (Brederoo, P. and De Priester, W., eds.), pp. 720–721) in combination with low temperature embedding in Lowicryl HM20 according to Humbel et al. (Humbel, B. and Müller M. (1984) in Proc. 8th Eur. Congr. on Electron Microscopy (Csanady, P., Röhlich, P.; Szabo D., eds.), pp. 1789–1798). With this method one can visualize a honeycomb structure with local fusion (joining) points between bilayers which is compatible with the structures hypothesized for lipidic particle-containing systems.     

Development of Epidermal Cell Wall Peroxidases along the Mung Bean Hypocotyl: Possible Involvement in the Cell Wall Stiffening Process

Goldberg, R., Liberman, M., Mathieu, C, Pierron, M. and Catesson,A. M. 1987. Development of epidermal cell wall peroxidases alongthe mung bean hypocotyl: possible involvement in the cell wallstiffening process.—J. exp. Bot. 38: 1378–1390. Ultrastructural investigation showed that in the epidermis ofmung bean hypocotyls, cell wall peroxidatic activities couldbe detected mainly below the maximal elongation zone. In theepidermis the peroxidatic activities were preferentially locatedin the radial cell walls. Cell wall peroxidases were then isolatedfrom epidermal strips and further characterized. The possiblepresence of a H₂O₂-generating system in the epidermis of mungbean hypocotyls was also investigated. When whole segments wereprocessed for electron microscopy, H₂O₂ could be detected cytochemicallyin the cell walls with the CeCl₃ technique. A positive reactionwas obtained in the same location when specimens were incubatedin a 3-3'-diaminobenzidine medium for peroxidases in which H₂O₂was replaced by its possible precursors (NADH or NAD + malate).However, isolated epidermal cell walls could not generate H₂O₂at the expense of NADH although they were able to oxidize thereduced nicotinamide-adenine-dinucleotide. The possible relationshipsbetween peroxidase activities, H₂O₂, and Ca²⁺ ions are discussedwith respect to their involvement in the cell wall stiffeningprocess. Key words: Epidermis, cell wall, elongation, peroxidases     

Restriction fragment length polymorphism (RFLP) analysis provides evidence for a high degree of homology of mitochondrial DNAs from rat hepatomasVersus normal rat livers

Where differences have been reported between tumor and normal mitochondrial DNA (mtDNA), they have generally involved limited modifications of the genome (Taira et al., Nucleic Acids Res. 11:1635, 1983; Shay and Werbin, Mutat. Res. 186:149, 1987). However, Corral et al. (Nucleic Acids Res. 16:10935, 1988; 17:5191, 1989) observed recombination between cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6), two genes normally on opposite sides of the circular mitochondrial genome. In rat hepatoma mtDNA COI and ND6 were reported to be separated by only 230 base pairs (Corral et al., 1988, 1989). We have performed RFLP analysis on mtDNA from normal rat livers and rat hepatomas, using COI and ND6 probes. Additional experiments compared end-labeled DNA fragments produced by EcoRI and HindIII digestion of mtDNA. These studies failed to provide any evidence for genetic recombination in rat hepatoma mtDNA, even in the same cell line used by Corral et al. Rather, they support the conclusion that mtDNA from tumor and normal tissues exhibits a low degree of heterogeneity.     

Establishing the triplet nature of the genetic code

In 1961, Crick, Barnett, Brenner, and Watts-Tobin (Crick et al., 1961) designed an elegant experimental strategy to determine the nature of the genetic code. Remarkably, they reached the correct conclusion despite the absence of technology to analyze and compare DNA and protein sequences.     

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequence analysis of porcine myoglobin cDNA

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.     

Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases.

Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.     

Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata

Catalase-peroxidase was isolated from aerobically grown Rhodopseudomonas capsulata. The enzyme resembles typical catalases in some of its physicochemical properties. It has an apparent molecular weight of 236,000 and is composed of four identical subunits. It shows a typical high spin ferric heme spectrum with absorption maxima at 403 and 635 nm and shoulders at 503 and 535 nm. Upon binding of cyanide, the enzyme is converted to the low spin state, as shown by the shift of the Soret maximum to 418 nm and the band at 532 nm. It has an isoelectric point at pH 4.5. The enzyme differs from typical catalases in also having a strong peroxidatic activity with dianisidine, pyrogallol, and diaminobenzidine as electron donors. Both the catalatic and the peroxidatic activities are similarly inactivated by treatment with 1 mM H2O2, heating to 50 degrees C, exposure to ethanol/chloroform, and photooxidative conditions. In contrast to typical catalases, but similarly to peroxidases, the enzyme is reduced by sodium dithionite. The pH optimum of the peroxidatic activity is 5-5.3 (in contrast to 6-6.5 of the catalatic activity). 50% of the apparent maximal activities are reached at 0.3 and 4.2 mM H2O2 for the peroxidatic and catalatic activities, respectively. Both enzymic activities are equally inhibited by cyanide, 50% inhibition being achieved with 2.2 X 10(-5) M KCN. Contrarily, the two activities differ in their response to hydroxylamine and azide. 50% inhibition of the catalatic activity is obtained with 1.5 X 10(-4) M azide or 2.15 X 10(-6) M hydroxylamine; 50% inhibition of the peroxidatic activity requires 7.3 X 10(-4) M azide or 7.8 X 10(-5) M hydroxylamine. The activation energies of the catalatic and the peroxidatic activities are 1.9 and 1.7 kcal/mol, respectively.     

Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: a mutagenesis study

Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c(551). Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a "charge hopping" mechanism may be operative in the process of intra-molecular electron transfer.     

Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases

The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103]. It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.     

Karyology and reproductive biology of Muscari matritensis M. Ruíz Rejón et al. (Liliaceae)

VALDÉS, B. & DÍAZ LIFANTE, Z., 1992. Karyology and reproductive biology of Muscari matritensis M. Ruíz Rejón et al. (Liliaceae). Muscari matritensis M. Ruíz Rejón et al. , previously known from only five localities, has been recorded for S.W. Spain. The plants have a karyotype composition which differs from the other four karyotypes described for this species, and experimental studies of the breeding system provide evidence that they are moderately autogamous.     

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.     

Relationship between O2 evolution capacity and cytochrome b-559 high-potential form in Photosystem II particles

As previously shown for inside-out vesicles by Larsson et al. (Larsson, C., Jansson, C., Ljungberg, U.L., Åkerlund, H.E. and Anderson, B. (1984) in Advances in Photosynthesis Research, Vol. I, pp. 363–366 (Sybesma C., ed.), Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, The Netherlands), we observed that NaCl 1 M washing of Photosystem II particles prepared by Triton X-100 treatment of spinach thylakoids induces both an inactivation of oxygen evolution and transformation of cytochrome b-559 from its high-potential to its low-potential form. A partial reactivation of water oxidation by 24 kDa polypeptide refixation is accompanied by a partial restoration of the cytochrome b-559 high-potential (HP) form. In contrast, reconstitution of water splitting by Ca²⁺ addition is not associated to a reestablishment of the cytochrome (HP) form. We conclude that cytochrome b-559 HP plays no role in water oxidation.     

Control of cytochrome oxidase activity. A transient spectroscopy study.

The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.     

Relation between interface properties and kinetics of electron transfer in the interaction of cytochrome f and plastocyanin from plants and the cyanobacterium Phormidium laminosum

Cytochrome f and plastocyanin from the cyanobacterium Phormidium laminosum react an order of magnitude faster than their counterparts from chloroplasts when long-range electrostatic interactions have been screened out by high salt concentration [Schlarb-Ridley, B. G., et al. (2002) Biochemistry 41, 3279-3285]. To investigate the relative contributions of the reaction partners to these differences, the reactions of turnip cytochrome f with P. laminosum plastocyanin and P. laminosum cytochrome f with pea plastocyanin were examined. Exchanging one of the plant reaction partners with the corresponding cyanobacterial protein nearly abolished electron transfer at low ionic strength but increased the rate at high ionic strength. This increase was larger for P. laminosum cytochrome f than for P. laminosumplastocyanin. To identify molecular features of P. laminosum cytochrome f that contribute to the increase, the effect of mutations in the N-terminal heme-shielding peptide on the reaction with P. laminosum plastocyanin was determined. Phenylalanine-3 was converted to valine and tryptophan-4 to phenylalanine or leucine. The mutations lowered the rate constant at 0.1 M ionic strength by factors of 0.71 for F4V, 0.42 for W4F, and 0.63 for W4L while introducing little change in the shape of the ionic strength dependence curve. When the N-terminal tetrapeptide (sequence YPFW) was converted into that found in the chloroplast of Chlamydomonas reinhardtii (YPVF), the reaction was slowed further (factor of 0.26). The N-terminal heme-shielding peptide was found to be responsible for 75% of the kinetic differences between cytochrome f from chloroplasts and the cyanobacterium when electrostatic interactions were eliminated.