Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.     

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Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of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.     

Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterologous synthesis of cytochrome c' by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery

Hydrogenophilus thermoluteolus cytochrome c' (PHCP) has typical spectral properties previously observed for other cytochromes c', which comprise Ambler's class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c' from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler's class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b(562), a 106-residue four-helix bundle.     

Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Site-directed mutagenesis of yeast cytochrome c peroxidase shows histidine 181 is not required for oxidation of ferrocytochrome c

The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution crystal structures and spectroscopy of native and compound I cytochrome c peroxidase

Cytochrome c peroxidase (CCP) is a 32.5 kDa mitochondrial intermembrane space heme peroxidase from Saccharomyces cerevisiae that reduces H(2)O(2) to 2H(2)O by oxidizing two molecules of cytochrome c (cyt c). Here we compare the 1.2 A native structure (CCP) with the 1.3 A structure of its stable oxidized reaction intermediate, Compound I (CCP1). In addition, crystals were analyzed by UV-vis absorption and electron paramagnetic resonance spectroscopies before and after data collection to determine the state of the Fe(IV) center and the cationic Trp191 radical formed in Compound I. The results show that X-ray exposure does not lead to reduction of Fe(IV) and only partial reduction of the Trp radical. A comparison of the two structures reveals subtle but important conformational changes that aid in the stabilization of the Trp191 cationic radical in Compound I. The higher-resolution data also enable a more accurate determination of changes in heme parameters. Most importantly, when one goes from resting state Fe(III) to Compound I, the His-Fe bond distance increases, the iron moves into the porphyrin plane leading to shorter pyrrole N-Fe bonds, and the Fe(IV)-O bond distance is 1.87 A, suggesting a single Fe(IV)-O bond and not the generally accepted double bond.     

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.     

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.     

Cytochrome c peroxidase compound ES is identical with horseradish peroxide compound I in iron-ligand distances

X-ray absorption studies of compound ES of cytochrome c peroxidase show a short iron-oxygen distance of 1.67 +/- 0.04 A, an iron-histamine distance of 1.91 +/- 0.03 A, and an iron-pyrrole nitrogen average distance of 2.02 +/- 0.02 A. This is identical within the error with the reported structure of horseradish peroxidase compound I [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611]. Comparisons of the structures of myoglobin peroxide [Chance, M., Powers, L., Kumar, C., & Chance, B. (1986) Biochemistry (preceding paper in this issue)], compound ES, and the intermediates of horseradish peroxidase reveal the possible mechanisms for the stabilization of the free radical species generated during catalysis. The proximal histidine regulates the structure and function of the pyrrole nitrogens and the heme, allowing for the formation and maintenance of the characteristic intermediates.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.     

Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase

A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix

The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength. The pH dependence of the reaction between CcP and peracetic acid has also been determined. The rate of the reactions are influenced by two heme-linked ionizations in the protein. The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated. The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively. These rates are about 60% slower than the reaction between hydrogen peroxide and CcP. A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme. The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids. These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions. However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion. The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.     

Cytochrome c peroxidase compound I: formation of covalent protein crosslinks during the endogenous reduction of the active site

Cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) was oxidized by hydrogen peroxide in the absence of exogenous electron donor. Higher molecular weight species were observed in the decay products at pH 4.5. Monomer and dimer were separated by gel filtration and purified by anion-exchange chromatography. Peptide mapping of tryptic digests of the dimer indicated a tyrosine crosslink localized between residues 32 and 48 of the native 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.     

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.     

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Abstract

Singlet oxygen (¹O₂) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe³⁺/O₂) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.