Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain.

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 ± 1) × 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study.

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

Pressure perturbation calorimetry, heat capacity and the role of water in protein stability and interactions.

It is widely acknowledged, and usually self-evident, that solvent water plays a crucial role in the overall thermodynamics of protein stabilization and biomolecular interactions. Yet we lack experimental techniques that can probe unambiguously the nature of protein-water or ligand-water interactions and how they might change during protein folding or ligand binding. PPC (pressure perturbation calorimetry) is a relatively new technique based on detection of the heat effects arising from application of relatively small pressure perturbations (+/-5 atm; 1 atm=101.325 kPa) to dilute aqueous solutions of proteins or other biomolecules. We show here how this can be related to changes in solvation/hydration during protein-protein and protein-ligand interactions. Measurements of 'anomalous' heat capacity effects in a wide variety of biomolecular interactions can also be related to solvation effects as part of a quite fundamental principle that is emerging, showing how the apparently unusual thermodynamics of interactions in water can be rationalized as an inevitable consequence of processes involving the co-operative interaction of multiple weak interactions. This leads to a generic picture of the thermodynamics of protein folding stabilization in which hydrogen-bonding plays a much more prominent role than has been hitherto supposed.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer.

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E (0)', of 230 +/- 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 +/- 0.2 x 10(5) M(-1) s(-1) was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus.

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

The structure of an electron transfer complex containing a cytochrome c and a peroxidase.

Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.

The surface-charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans--implications for the interaction with cytochrome c peroxidase.

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H-NMR spectroscopy shows that it is the monomer that binds.

Structural characterization of Paracoccus denitrificans cytochrome c peroxidase and assignment of the low and high potential heme sites.

The amino acid sequence of the diheme cytochrome c peroxidase from Paracoccus denitrificans has been determined as the result of sequence analysis of peptides generated by chemical and enzymatic cleavages of the apoprotein. The sequence shows 60% similarity to the cytochrome c peroxidase from Pseudomonas aeruginosa, 39% similarity to an open reading frame encoding a putative triheme c-type cytochrome in Escherichia coli, and remote similarity to the MauG proteins from two methylotrophic bacteria. It is proposed, on the basis of the pattern of conserved residues in the sequences, that a change in iron coordination in the N-terminal heme domain may accompany reduction to the active mixed valence state, a change which may be accompanied by conformational adjustments in the highly conserved interface between the N- and C-terminal domains. These conformational adjustments may also lead to the appearance of a second Ca2+ binding site in the mixed valence enzyme. The exposed edge of the heme in the C-terminal domain is surrounded by several different patterns of charged residues in the Paracoccus and Pseudomonas enzymes, and this is consistent with the interaction of the former with the highly positively charged front face of the donor cytochrome c-550.

A single histidine is required for activity of cytochrome c peroxidase from Paracoccus denitrificans.

The diheme cytochrome c peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was modified in the oxidized enzyme, and modification was associated with loss of the ability to form the active state. With time, the modification reversed, and the ability to form the active state was recovered. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. However, the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity, and we propose a slow transfer of the ethoxyformyl group to an unidentified amino acid. The presence of CN- bound to the active peroxidatic site of the enzyme led to complete protection of the essential histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state.

The affinity and specificity of Ca(2+)-binding sites of cytochrome-c peroxidase from Paracoccus denitrificans.

The binding of Ca2+ to the dihaem cytochrome-c peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and 1H-NMR spectra of both haem groups. The enzyme contains at least two types of Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM and accommodates neither Mg2+ nor Mn2+. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca2+ cooperatively with a Kd of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by -Ca2+ binding to site II, the titration of haem methyl 1H-NMR resonances, and a half-of-sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca2+ affinity. Binding of Ca2+ to site II is required for formation of the active high spin state at the peroxidatic haem.

Mössbauer characterization of Paracoccus denitrificans cytochrome c peroxidase. Further evidence for redox and calcium binding-induced heme-heme interaction.

Mössbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mössbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S = 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with gmax = 2.89 (species I) and gmax = 2.78 (species II). This EPR observation, together with the Mössbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.

The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase.

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.

Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans.

The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.

Cytochrome c' of Paracoccus denitrificans.

Cytochrome c' was identified in periplasmic extracts of the Paracoccus denitrificans strains LMD 22.21 and LMD 52.44. The cytochrome c' was purified from the latter using the device of sequential molecular exclusion chromatography in the dimeric and monomeric states. Although showing the overall spectroscopic features of the cytochrome c' family, the Paracoccus cytochrome c' is unusual in having a red-shifted oxidised Soret band at 407 nm. Also unusual is the midpoint potential of 202 mV, well above the known cytochrome c' range. The amino-acid composition of Pa. denitrificans cytochrome c' showed the high alanine and low proline content characteristic of the group and reflecting the predominantly alpha-helical character of the protein. Comparison of the amino-acid compositions suggests some similarity to the cytochromes c' of Chromatium vinosum and halotolerant Paracoccus.

The cytochrome c peroxidase of Paracoccus denitrificans.

The size, visible absorption spectra, nature of haem and haem content suggest that the cytochrome c peroxidase of Paracoccus denitrificans is related to that of Pseudomonas aeruginosa. However, the Paracoccus enzyme shows a preference for cytochrome c donors with a positively charged 'front surface' and in this respect resembles the cytochrome c peroxidase from Saccharomyces cerevisiae. Paracoccus cytochrome c-550 is the best electron donor tested and, in spite of an acidic isoelectric point, has a markedly asymmetric charge distribution with a strongly positive 'front face'. Mitochondrial cytochromes c have a much less pronounced charge asymmetry but are basic overall. This difference between cytochrome c-550 and mitochondrial cytochrome c may reflect subtle differences in their electron transport roles. A dendrogram of cytochrome c1 sequences shows that Rhodopseudomonas viridis is a closer relative of mitochondria than is Pa. denitrificans. Perhaps a mitochondrial-type cytochrome c peroxidase may be found in such an organism.

The cellular location and specificity of bacterial cytochrome c peroxidases.

The locations of cytochrome c peroxidase and catalase activities in the two Gram-negative bacteria Pseudomonas stutzeri (N.C.I.B. 9721) and Paracoccus denitrificans (N.C.I.B. 8944) were investigated by the production of spheroplasts. In both species the cytochrome c peroxidase was predominantly periplasmic: 92% of total activity in Ps. stutzeri and 98% of nonmembrane-bound activity in Pa. denitrificans were found in this cellular compartment. In contrast, the catalase was mostly in the cytoplasmic fraction. Purification of the Pa. denitrificans cytochrome c peroxidase showed it to be the haem c-containing polypeptide of Mr 42,000 that has already been described by Bosma, Braster, Stouthamer & Van Versefeld [(1987) Eur. J. Biochem. 165, 665-670] but was not identified by them as a peroxidase. The visible-absorption spectra of the enzyme closely resemble those of cytochrome c peroxidase from Pseudomonas aeruginosa but the donor specificity is different, with the Pa. denitrificans enzyme preferring the basic mitochondrial cytochromes c to the acidic cytochromes c-551 and reacting well with the Pa. denitrificans cytochrome c-550.

The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes.

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN'N'-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.

Free and membrane-bound forms of bacterial cytochrome c4.

Cytochrome c4 was isolated from cells of Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii. The dihaem nature, Mr of approx. 20,000 and ferrohaem spectra in the region of the alpha- and beta-peaks define this family of cytochromes c. The behaviour of the holocytochromes in SDS was atypical, but removal of the haem groups resulted in a normal migration. In all three organisms most of the cytochrome c4 was tightly bound to the membrane, but some free cytochrome was detected. The membrane-attached cytochrome could be extracted with butanol, and this solubilized form was then indistinguishable in properties from the free form. Denitrifying rather than aerobic growth conditions hardly affected the total cytochrome c4 in the two pseudomonads, but there was slightly more free form and less membrane-attached form in denitrifying growth. The nature of the attachment of cytochrome c4 to the membrane is discussed and a model is proposed for the process of solubilization.