A genetic toolbox to empower Paracoccus pantotrophus DSM 2944 as a metabolically versatile SynBio chassis.

BACKGROUND: To contribute to the discovery of new microbial strains with metabolic and physiological robustness and develop them into successful chasses, Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium from the phylum Alphaproteobacteria and the family Rhodobacteraceae, was chosen. The strain possesses an innate ability to tolerate high salt concentrations. It utilizes diverse substrates, including cheap and renewable feedstocks, such as C1 and C2 compounds. Also, it can consume short-chain alkanes, predominately found in hydrocarbon-rich environments, making it a potential bioremediation agent. The demonstrated metabolic versatility, coupled with the synthesis of the biodegradable polymer polyhydroxyalkanoate, positions this microbial strain as a noteworthy candidate for advancing the principles of a circular bioeconomy. RESULTS: The study aims to follow the chassis roadmap, as depicted by Calero and Nikel, and de Lorenzo, to transform wild-type P. pantotrophus DSM 2944 into a proficient SynBio (Synthetic Biology) chassis. The initial findings highlight the antibiotic resistance profile of this prospective SynBio chassis. Subsequently, the best origin of replication (ori) was identified as RK2. In contrast, the non-replicative ori R6K was selected for the development of a suicide plasmid necessary for genome integration or gene deletion. Moreover, when assessing the most effective method for gene transfer, it was observed that conjugation had superior efficiency compared to electroporation, while transformation by heat shock was ineffective. Robust host fitness was demonstrated by stable plasmid maintenance, while standardized gene expression using an array of synthetic promoters could be shown. pEMG-based scarless gene deletion was successfully adapted, allowing gene deletion and integration. The successful integration of a gene cassette for terephthalic acid degradation is showcased. The resulting strain can grow on both monomers of polyethylene terephthalate (PET), with an increased growth rate achieved through adaptive laboratory evolution. CONCLUSION: The chassis roadmap for the development of P. pantotrophus DSM 2944 into a proficient SynBio chassis was implemented. The presented genetic toolkit allows genome editing and therewith the possibility to exploit Paracoccus for a myriad of applications.

Performance of Paracoccus pantotrophus MA3 in heterotrophic nitrification-anaerobic denitrification using formic acid as a carbon source.

Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

Eco-friendly decolorization and degradation of reactive yellow 145 textile dye by Pseudomonas aeruginosa and Thiosphaera pantotropha.

Dyes are toxic and inherently resistant to microbial degradation. In this study, decolorization and degradation of textile dye reactive yellow 145 (RY145) were evaluated using pure bacterial strains Pseudomonas aeruginosa (RS1) and Thiosphaera pantotropha ATCC 35512. In nutrient broth under static condition, complete decolorization of 50 mg L-1 RY145 could be achieved within 96 h and 72 h, for Pseudomonas aeruginosa (RS1) and Thiosphaera pantotropha, respectively. In contrast, under shaking condition both the cultures could achieve only 50% decolorization in 96 h. Treatment under sequential static and shaking condition resulted in complete decolorization and 65% mineralization after 96 h. Higher dye concentration in excess of 100 mg L-1 and 50 mg L-1 decreased the extent of dye mineralization in Pseudomonas aeruginosa and Thiosphaera pantotropha, respectively. Even with the repetitive addition of the dye, both the strains were capable of decolorizing the dye. Acclimatized cultures showed 54% decolorization of RY145 in mineral media (MM) even in the absence of a readily degradable external carbon source. Amongst various individual carbon and nitrogen sources, maximum decolorization was observed in MM supplemented with peptone as carbon and nitrogen source at pH 7 under static condition.

Sustainable biological system for the removal of high strength ammoniacal nitrogen and organic pollutants in poultry waste processing industrial effluent.

The effluent generated from poultry waste processing industries contains several organic compounds such as collagen, gelatin, bovine serum albumin, carbohydrates, essential fatty acids, and so forth. This enabled the establishment of poultry waste processing industries to produce value-added products such as animal feed and organic fertilizers. During poultry waste processing, huge amounts of ammoniacal nitrogen and organic pollutants such as proteins, various carbohydrates, and fatty materials are discharged into the effluent stream which contributes to several environmental issues. Because of the shortcomings of the current conventional treatment, the present study is about with the development of a sequential bioreactor system for the effective treatment of poultry waste processing industrial effluent. Facultative anaerobe Paracoccus pantotrophus FMR19 along with the indigenous isolate Bacillus albus MN527241 obtained from clarifying sludge was used as mixed consortia for the treatment of poultry waste processing industrial effluent. The mixed microbial consortia resulted in the maximum activity of enzymes such as protease (247 U/mL) and lipase (28.266 U/mL) thereby achieving 90% of ammoniacal nitrogen reduction and 98% of COD removal within five days. Further, the confirmatory analysis of poultry effluent treatment was carried out using gas chromatography-mass spectroscopy (GC-MS), High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and SDS-PAGE. Hence, the sequential bioreactor-based treatment approach has proved to be highly effective in removal of organic pollutants in the poultry waste processing industrial effluent.Implications: The poultry waste processing industrial (PWPI) effluent contains huge ammoniacal nitrogen and COD and affects the environment. Aerobic moving bed biofilm reactor and up-flow anaerobic sludge blanket reactor are in current practice and shows considerable reduction in the ammoniacal nitrogen and COD in long retention time. Therefore, there is a need of sustainable treatment process that could effectively remove the organic pollutants from the effluent in short duration. Our study focused on the application of sequential bioreactor approach for the treatment of PWPI using aerobic followed by anaerobic treatment process and observed efficient organic pollutants removal in short duration.

Nitrate stimulation of N-Methylpyrrolidone biodegradation by Paracoccus pantotrophus: Metabolite mechanism and Genomic characterization.

Due to the toxicological nature of N-methylpyrrolidone (NMP), the conventional anaerobic bioprocess is quite ineffective for NMP removal from wastewater. In order to achieve effective NMP biodegradation under anoxic condition, Paracoccus pantotrophus NJUST38 was isolated for the first time. The supplementation of nitrate into anoxic system resulted in complete removal of 5 mM NMP by NJUST38 within 11 h compared to 24% in the anaerobic control system in the absence of nitrate. Genome characterization revealed that NMP biodegradation catalyzed by several key enzymes/genes, including N-methylhydantoin amidohydrolase (hyuB), methyltransferase (cobA), 4-aminobutyrate-2-oxoglutarate transaminase (gabT), succinate-semialdehyde dehydrogenase (gabD) and so on. NMP biodegradation pathway was proposed based on several intermediates, where NMP was biodegraded mainly for providing electrons and reducing power to support microbial denitrification through tricarboxylic acid (TCA) cycle. The proposed mechanism should aid our mechanistic understanding of NMP biodegradation by Paracoccus pantotrophus and the development of sustainable bioremediation strategies.

Comparison of sulphide and nitrate removal from synthetic wastewater by pure and mixed cultures of nitrate-reducing, sulphide-oxidizing bacteria.

In this study, the activities of hydrogen sulphide (H2S) oxidation and nitrate (N-NO3-) reduction by three pure and mixed strains of nitrate-reducing, sulphide oxidizing bacteria (NR-SOB) were determined. Batch experiments were performed at 35 °C and pH 7.0-8.0 with initial H2S concentrations of 650-900 ppmv and N-NO3- concentrations of ∼120 mg/L. The strains MAL 1HM19, TPN 1HM1 and TPN 3HM1 were capable of removing 100% gas-phase H2S. The co-cultures showed better performance for H2S and N-NO3- removal. The mixed NR-SOB strains showed a higher H2S oxidation rate (143 ±â€¯18 ppmv/h), while the highest N-NO3- removal rate (5.5 ±â€¯0 and 5.1 ±â€¯0.6 N-NO3- mg/L·h) was obtained by a mixture of two NR-SOB strains. The 16S rDNA sequence analysis revealed that all strains belonged to the sub-class Alphaproteobacteria and are closely related to Paracoccus sp. (>99%).

Recombinant Sox Enzymes from Paracoccus pantotrophus Degrade Hydrogen Sulfide, a Major Component of Oral Malodor.

Hydrogen sulfide (H2S) is emitted from industrial activities, and several chemotrophs possessing Sox enzymes are used for its removal. Oral malodor is a common issue in the dental field and major malodorous components are volatile sulfur compounds (VSCs), including H2S and methyl mercaptan. Paracoccus pantotrophus is an aerobic, neutrophilic facultatively autotrophic bacterium that possesses sulfur-oxidizing (Sox) enzymes in order to use sulfur compounds as an energy source. In the present study, we cloned the Sox enzymes of P. pantotrophus GB17 and evaluated their VSC-degrading activities for the prevention of oral malodor. Six genes, soxX, soxY, soxZ, soxA, soxB, and soxCD, were amplified from P. pantotrophus GB17. Each fragment was cloned into a vector for the expression of 6×His-tagged fusion proteins in Escherichia coli. Recombinant Sox (rSox) proteins were purified from whole-cell extracts of E. coli using nickel affinity chromatography. The enzyme mixture was investigated for the degradation of VSCs using gas chromatography. Each of the rSox enzymes was purified to apparent homogeneity, as confirmed by SDS-PAGE. The rSox enzyme mixture degraded H2S in dose- and time-dependent manners. All rSox enzymes were necessary for degrading H2S. The H2S-degrading activities of rSox enzymes were stable at 25-80°C, and the optimum pH was 7.0. The amount of H2S produced by periodontopathic bacteria or oral bacteria collected from human subjects decreased after an incubation with rSox enzymes. These results suggest that the combination of rSox enzymes from P. pantotrophus GB17 is useful for the prevention of oral malodor.

Start-up of sequencing batch reactor with Thiosphaera pantotropha for treatment of high-strength nitrogenous wastewater and sludge characterization.

Biological treatment of high-strength nitrogenous wastewater is challenging due to low growth rate of autotrophic nitrifiers. This study reports bioaugmentation of Thiosphaera pantotropha capable of simultaneously performing heterotrophic nitrification and aerobic denitrification (SND) in sequencing batch reactors (SBRs). SBRs fed with 1:1 organic-nitrogen (N) and NH4+-N were started up with activated sludge and T. pantotropha by gradual increase in N concentration. Sludge bulking problems initially observed could be overcome through improved aeration and mixing and change in carbon source. N removal decreased with increase in initial nitrogen concentration, and only 50-60 % removal could be achieved at the highest N concentration of 1000 mg L-1 at 12-h cycle time. SND accounted for 28 % nitrogen loss. Reducing the settling time to 5-10 min and addition of divalent metal ions gradually improved the settling characteristics of sludge. Sludge aggregates of 0.05-0.2 mm diameter, much smaller than typical aerobic granules, were formed and progressive increase in settling velocity, specific gravity, Ca2+, Mg2+, protein, and polysaccharides was observed over time. Granulation facilitated total nitrogen (TN) removal at a constant rate over the entire 12-h cycle and thus increased TN removal up to 70 %. Concentrations of NO2--N and NO3--N were consistently low indicating effective denitrification. Nitrogen removal was possibly limited by urea hydrolysis/nitrification. Presence of T. pantotropha in the SBRs was confirmed through biochemical tests and 16S rDNA analysis.

A novel application of Paracoccus pantotrophus for the decolorization of melanoidins from distillery effluent under static conditions.

Melanoidin is the hazardous byproduct formed during the production of ethanol in distilleries. In the present study, a highly effective melanoidin decolorizing bacterial isolate, SAG1, was isolated from the effluent enriched soil of a distillery. This strain, identified as Paracoccus pantotrophus, was highly efficient to decolorize melanoidins up to 81.2 ± 2.43% in the presence of glucose and NH4NO3. The effects of autoclaved as well as living cells and inoculums size on decolorization activity were investigated. The results indicated that only living cell showed the decolorization activity i.e. 78.6 ± 2.62%, while, no activity has been observed using autoclaved cells. The inoculums size of 8% v/v, showed maximum activity of 62.9 ± 3.00%. The isolate SAG1 was found to be more efficient in decolorizing the melanoidins from distillery effluent as compared to the reference culture Pseudomonas putida.

Sulphate production by Paracoccus pantotrophus ATCC 35512 from different sulphur substrates: sodium thiosulphate, sulphite and sulphide.

One of the problems in waste water treatment plants (WWTPs) is the increase in emissions of hydrogen sulphide (H2S), which can cause damage to the health of human populations and ecosystems. To control emissions of this gas, sulphur-oxidizing bacteria can be used to convert H2S to sulphate. In this work, sulphate detection was performed by spectrophotometry, ion chromatography and atomic absorption spectrometry, using Paracoccus pantotrophus ATCC 35512 as a reference strain growing in an inorganic broth supplemented with sodium thiosulphate (Na2S2O3·5H2O), sodium sulphide (Na2S) or sodium sulphite (Na2SO3), separately. The strain was metabolically competent in sulphate production. However, it was only possible to observe significant differences in sulphate production compared to abiotic control when the inorganic medium was supplemented with sodium thiosulphate. The three methods for sulphate detection showed similar patterns, although the chromatographic method was the most sensitive for this study. This strain can be used as a reference for sulphate production in studies with sulphur-oxidizing bacteria originating from environmental samples of WWTPs.

Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in α-proteobacteria.

It has recently been shown that the biosynthetic route for both the d1 -haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1 -haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.

Kinetic enrichment of 34S during proteobacterial thiosulfate oxidation and the conserved role of SoxB in S-S bond breaking.

During chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of (34)S in the end product sulfate, with overall fractionation ranging between -4.6‰ and +5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of (34)S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical (34)S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.

Nitrophenol removal by simultaneous nitrification denitrification (SND) using T. pantotropha in sequencing batch reactors (SBR).

Nitrophenol removal was assessed using four identical lab scale sequencing batch reactors R (background control), R1 (4-nitrophenol i.e. 4-NP), R2 (2,4-dinitrophenol i.e. 2,4-DNP), and R3 (2,4,6-trinitrophenol i.e. 2,4,6-TNP). In the present study, the SND based SBR system was used to carry out total nitrogen removal at reduced aeration (DO=2mg/L) using a specifically designed single sludge biomass containing Thiosphaera pantotropha. The concentration of each of the nitrophenols was gradually increased from 2.5 to 200mg/L during acclimation. The nitrophenols were used as the sole source of nitrogen during study. A synthetic feed was designed to direct SND in the bioreactors. It was observed that overall removal for 4-NP was 98% and for 2,4-DNP and 2,4,6 TNP, removals varied between 83% and 84%. The COD removal for 4-NP was 99% and for 2,4-DNP and 2,4,6-TNP was 97-98% during acclimation. Total nitrogen and nitrophenol removals were achieved via SND.

Analysis of resonance Raman data on the blue copper site in pseudoazurin: excited state π and σ charge transfer distortions and their relation to ground state reorganization energy.

The short Cu(2+)-S(Met) bond in pseudoazurin (PAz) results in the presence of two relatively intense S(p)(π) and S(p)(σ) charge transfer (CT) transitions. This has enabled resonance Raman (rR) data to be obtained for each excited state. The rR data show very different intensity distribution patterns for the vibrations in the 300-500 cm(-1) region. Time-dependent density functional theory (TDDFT) calculations have been used to determine that the change in intensity distribution between the S(p)(π) and S(p)(σ) excited states reflects the differential enhancement of S(Cys) backbone modes with Cu-S(Cys)-C(ß) out-of-plane (oop) and in-plane (ip) bend character in their respective potential energy distributions (PEDs). The rR excited state distortions have been related to ground state reorganization energies (λ s) and predict that, in addition to M-L stretches, the Cu-S(Cys)-C(ß) oop bend needs to be considered. DFT calculations predict a large distortion in the Cu-S(Cys)-C(ß) oop bending coordinate upon reduction of a blue copper (BC) site; however, this distortion is not present in the X-ray crystal structures of reduced BC sites. The lack of Cu-S(Cys)-C(ß) oop distortion upon reduction corresponds to a previously unconsidered constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites.

Efficient synthesis of a chiral precursor for angiotensin-converting enzyme (ACE) inhibitors in high space-time yield by a new reductase without external cofactors.

A new reductase, CgKR2, with the ability to reduce ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), an important chiral precursor for angiotensin-converting enzyme (ACE) inhibitors, was discovered. For the first time, (R)-HPBE with >99% ee was produced via bioreduction of OPBE at 1 M without external addition of cofactors. The space-time yield (700 g·L(-1)·d(-1)) was 27 times higher than the highest record.

Effect of shock and mixed loading on the performance of SND based sequencing batch reactors (SBR) degrading nitrophenols.

The effect of nitrophenolic shock loads on the performance of three lab scale SBRs was studied using a synthetic feed. Nitrophenols were biotransformed by Simultaneous heterotrophic Nitrification and aerobic Denitrification (SND) using a specially designed single sludge biomass containing Thiosphaera pantotropha. Reactors R1, R2 and R3 were fed with 200mg/L concentration of 4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP), and 2,4,6-trinitrophenol (2,4,6-TNP) whereas reactor R was used as a background control. Three nitrophenolic shock loadings of 400, 600 and 800 mg/Ld were administrated by increasing the influent nitrophenolic concentration while keeping the hydraulic retention time as 48 h. The shocks were given continuously for a period of 4 days before switching back to normal nitrophenolic loading (200mg/Ld). The reactors were allowed to recover to normal performance level before administrating the next nitrophenolic shock load. The study showed that a nitrophenolic shock load, as high as 600 mg/Ld was completely degraded by the 4-NP & 2,4-DNP bioreactors while almost half degraded by the 2,4,6-TNP bioreactor without affecting the reactor's performance irreversibly. After resuming the normal nitrophenolic loading, it took almost 8-10 days for the reactors to recover from the shock effect. The study was further extended to evaluate the maximum possible mixed nitrophenolic loading (4-NP:2,4-DNP:2,4,6-TNP 1:1:1) to which a reactor (R3) containing 2,4,6-TNP acclimated single sludge biomass can be exposed without hampering the reactor performance irreversibly. The reactor was able to achieve pseudo-steady-state at a mixed nitrophenolic loading of 300 mg/Ld with more than 90% removal of all the three nitrophenols, but could remove half of the mixed nitrophenolic loading of 600 mg/Ld.

Isolation, identification and removal of filamentous organism from SND based SBR degrading nitrophenols.

Four identical lab scale sequencing batch reactors R, R1, R2, and R3, were used to assess nitrophenol biodegradation using a single sludge biomass containing Thiosphaera pantotropha. Nitrophenols [4-Nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP) and 2,4,6-trinitrophenol (2,4,6-TNP)] were biotransformed by heterotrophic nitrification and aerobic denitrification (SND). Reactor R was used as background control, whereas R1, R2, and R3 were fed with 4-NP, 2,4-DNP, and 2,4,6-TNP, respectively. The concentration of each nitrophenol was gradually increased from 2.5 to 200 mg/l along with increase in COD, during acclimation studies. The final COD maintained was 4,500 mg/l with each nitrophenolic loading of 200 mg/l. During late phase of acclimation and HRT study, a filamentous organism started appearing in 2,4-DNP and 2,4,6-TNP bioreactors. Filaments were never found in 4-NP and background control reactor. Biochemistry and physiology behind filamentous organism development, was studied to obtain permanent solution for its removal. The effect of different input parameters such as COD loading, DO levels, SVI etc. were analyzed. The morphology and development of filamentous organism were examined extensively using microscopic techniques involving ESEM, oil immersion, phase contrast, and dark field microscopy. The organism was grown and isolated on selective agar plates and was identified as member of Streptomyses species.

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy.

Magnetic circular dichroism (MCD) spectra, at ultraviolet-visible or near-infrared wavelengths (185-2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.

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.

The relationship between redox enzyme activity and electrochemical potential-cellular and mechanistic implications from protein film electrochemistry.

In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies.