Synthesis of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Methanol and n-Amyl Alcohol by the Methylotrophic Bacteria Paracoccus denitrificans and Methylobacterium extorquens

Strains of two types of methylotrophic bacteria, Paracoccus denitrificans and Methylobacterium extorquens, synthesized the copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when methanol and n-amyl alcohol were added together to nitrogen-limited medium. The composition of the copolyester differed considerably between the two strains: the copolyester from P. denitrificans was comparatively rich in 3-hydroxyvalerate (3HV). The 3HV content of the copolyester synthesized by this strain increased with increasing concentrations of n-amyl alcohol. Its maximum content was 91.5 mol% under the conditions used. In M. extorquens, the maximum 3HV content was limited to 38.2 mol%. Since n-amyl alcohol served as a substrate for a standard methanol dehydrogenase, the enzyme was proposed to oxidize both methanol and n-amyl alcohol in the first step of copolyester synthesis from these substrates by methanol-grown cells.     

补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究

对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3－羟基丁酸和3－羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸（3HV前体）策略进行了研究,在6．6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149．9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12．4mol%),生产强度达到2．50（g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160．2g/L,119.0g/L,74.2%（其中3HV组分占PHA的17．7mol%)生产强度达到2．64（g.h^-1.L^-1)。     

High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Poly(3-Hydroxyvalerate) Depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA_SCL) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596–607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA_SCL depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M_r], 43,610 Da) resembles precursors of other extracellular PHA_SCL depolymerases (28 to 50% identical amino acids). The mature protein (M_r, 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S₁₃₆, D₂₁₁, and H₂₆₉ similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA_SCL depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA_SCL depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA_SCL depolymerases.     

Production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans containing aminotransferase activity

Summary To establish an efficient production method for l-phenylalanine, the production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans pFPr-1 containing aminotransferase activity was investigated. By using intact cells, 0.74M l-phenylalanine was produced from 0.8M phenylpyruvate (conversion yield, 92.5%). Moreover, by using immobilized cells with -carrageenan, when the space velocity was 0.1 h^-1 at 30°C, 0.135 M l-phenylalanine was produced from 0.15 M phenylpyruvate (conversion yield, 90%). The half-life of the l-phenylalanine-forming activity of the column was estimated to be about 30 days at 30°C.     

Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans.

A 3.6-kb EcoRI-SalI fragment of Paracoccus denitrificans DNA hybridized with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthase gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis of this region showed the presence of a 1,872-bp open reading frame (ORF), which corresponded to a polypeptide with a molecular weight of 69,537. Upstream of the ORF, a promoter-like sequence was found. Escherichia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of beta-ketothiolase and acetoacetylcoenzyme A reductase genes of A. eutrophus. The ORF was designated phaCPd. A plasmid vector carrying the phaCPd'-'lacZ fusion gene downstream of the promoter-like sequence expressed beta-galactosidase activity in P. denitrificans. When a multicopy and broad-host-range vector carrying the ORF along with the promoter-like sequence was introduced into P. denitrificans, the PHA content in the cells increased by twofold compared with cells carrying only a vector sequence.     

Biosynthesis of Poly(3-Hydroxybutyrate) and Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) and Its Regulation in Bacteria

Recent data on the biosynthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and its regulation in bacteria are reviewed, with special emphasis on the properties and regulation of the relevant enzymes and their genes. Some conditions promoting the synthesis of PHB and PHBV by natural, mutant, and recombinant producers are considered.     

Poly(3-Hydroxybutyrate) Production from Glycerol by Zobellella denitrificans MW1 via High-Cell-Density Fed-Batch Fermentation and Simplified Solvent Extraction

Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by high production costs, among which the costs for substrates and for downstream processing represent the main obstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of the biodiesel industry, have emerged to be promising carbon sources for industrial fermentations. In this study, Zobellella denitrificans MW1, a recently isolated bacterium, was used for the production of poly(3-hydroxybutyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conducted to scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relatively high cell density (29.9 ± 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, the PHB content was relatively low (31.0% ± 4.2% [wt/wt]). Afterwards, much higher concentrations of PHB (up to 54.3 ± 7.9 g/liter) and higher cell densities (up to 81.2 ± 2.5 g/liter) were obtained by further fed-batch optimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a short doubling time (1.64 h) were attained. The maximum PHB content obtained was 66.9% ± 7.6% of cell dry weight, and the maximum polymer productivity and substrate yield coefficient were 1.09 ± 0.16 g/liter/h and 0.25 ± 0.04 g PHB/g glycerol, respectively. Furthermore, a simple organic solvent extraction process was employed for PHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chloroform allowed a convenient separation of a clear PHB-solvent solution from the cells. Maximum PHB recovery (85.0% ± 0.10% [wt/wt]) was reached after 72 h of extraction with chloroform at 30°C, with a polymer purity of 98.3% ± 1.3%.Polyhydroxybutyrate (PHB) is the best-studied example of biodegradable polyesters belonging to the group of polyhydroxyalkanoates (PHAs), which are synthesized by many bacteria and archaea as intracellular carbon and energy reserves (1, 40, 43). In the last decades, these biopolymers have received great attention due to their properties which resemble those of conventional petrochemical-based polymers (49). For instance, PHB is very similar to thermoplastic polypropylene (17). Their production from renewable resources and their complete biodegradability give PHAs promising advantages from an environmental point of view (6). In addition to their special physical traits, such as the elasticity of medium-chain-length PHAs and the high crystallization rate of PHB, PHAs are biocompatible, water resistant, oxygen impermeable, and enantiomerically pure; all of these characteristics broaden the scope of their applications in industry and medicine.So far, higher production costs than those of petrochemical plastics have hindered the successful commercialization of PHB (9). Many efforts have been devoted to reducing the production costs by developing superior microbial strains capable of utilizing cheap substrates and also by applying more efficient fermentation strategies and economical recovery processes (10).Fed-batch fermentation regimens are usually applied to achieve a high cell density, which is necessary for a high productivity and yield, in particular in cases of intracellular products, by frequent or continuous feeding of nutrients when growth proceeds (46). Several fed-batch fermentation processes have been reported for PHA production (21, 28). There are two prevalent cultivation modes for PHB production that are imposed on the microorganisms being used. The more frequently used mode is realized by a complex two-stage cultivation process. In this mode, all nutrients needed for growth to a high cell density are provided during the first phase of the process. In the second phase, imbalanced growth conditions are enforced by providing growth-limiting amounts of nutrients such as nitrogen, phosphate, or oxygen to trigger PHA biosynthesis and accumulation. The model organism for this mode is Ralstonia eutropha (formerly known as Alcaligenes eutrophus and recently reclassified as Cupriavidus necator) (26, 27). In the other cultivation mode, PHB is accumulated concurrently with growth, and therefore a single-stage process is applicable. A well-known example of this mode is PHB production by Alcaligenes latus (18, 47).Although several new downstream processes for the extraction of PHA have been reported as being economically effective, such as the application of surfactants and hypochlorite (9, 38), dispersions of hypochlorite solution and chloroform (14, 15), and the selective dissolution of cell mass by proteolytic enzymes (25) or by sulfuric acid and hypochlorite (48), solvent extraction methods are still regarded as an adequate way to gain intact polymers with a high purity and recovery yield (39). However, there is still a need to develop and improve these extraction methods further to make the entire process much simpler and cheaper (22).In addition to increased prices for crude oil, the abundance of inexpensive raw materials from agriculture and industry as cheap substrates for microbial fermentations, such as crude glycerol from the biodiesel industry, could make the production of PHA from renewable resources more competitive with common plastics (32). Due to increased glycerol production by the growing biodiesel industry, the prices for glycerol became low enough to make this residual compound a cheap carbon source for several industrial fermentation processes, especially for the production of microbial polyesters (11, 34). However, the various amounts of actual fermentable substrates and the presence of other nonfermentable components in feedstock, such as the various concentrations of glycerol and salts in biodiesel coproducts, hinder their use (42). Therefore, tolerant bioprocesses and/or strains tolerant to such variable factors are required.The production of PHA from glycerol has been investigated in only a few studies (4, 12, 24, 33, 42). In a recent study (32), crude glycerol from different biodiesel manufacturers was examined for suitability as a substrate for PHB production. However, significant decreases in PHB productivity and product yields were recorded when NaCl-contaminated crude glycerol was used.Recently, a newly isolated bacterium, Zobellella denitrificans MW1, was characterized as producing large amounts of PHB from glycerol, with enhanced growth and polymer productivity in the presence of NaCl (20). The present study aimed at developing a strategy to improve the volumetric production of PHB by Z. denitrificans MW1, using glycerol as the sole carbon source. For this purpose, several fed-batch cultivations were set up to steadily improve the nutrient supply to attain a high cell density and high PHB productivity. Moreover, the conventional organic solvent extraction method was modified with regard to an economically more feasible large-scale PHB extraction, achieving a high purity and recovery of the polymer.     

Biodegradation of Isopropanol by a Solvent-Tolerant Paracoccus denitrificans Strain

The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5 × 10⁸ cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (μ _max ) = 0.0277/hr, a saturation constant (K _S ) = 0.7333 g/L, and an inhibition concentration (Ki) = 8.9887 g/L. Paracoccus denitrificans GH3 is considered to be well used in degrading IPA in wastewater.     

Metabolic Engineering of a Novel Propionate-Independent Pathway for the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) in Recombinant Salmonella enterica Serovar Typhimurium

A pathway was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a prpC strain of Salmonella enterica serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the Acinetobacter polyhydroxyalkanoate synthesis operon (phaBCA) and a second plasmid with the Escherichia coli sbm and ygfG genes under an independent promoter. The sbm and ygfG genes encode a novel (2R)-methylmalonyl-CoA mutase and a (2R)-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The S. enterica system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B₁₂) concentration in the medium.     

Complete Genome Sequence of the Metabolically Versatile Halophilic Archaeon Haloferax mediterranei, a Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Producer

Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids.     

Effects of Low Dissolved-Oxygen Concentrations on Poly-(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Production by Alcaligenes eutrophus

The bacterial copolyester poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced with Alcaligenes eutrophus DSM 545 from glucose and sodium propionate in a fed-batch fermentation with both nitrogen limitation and low dissolved-oxygen concentrations. When the dissolved-oxygen content was kept between 1 and 4% of air saturation during the polymer accumulation phase, the yield of 3-hydroxybutyrate (3HB) monomer from glucose was not affected, but the propionate-to-3-hydroxyvalerate (3HV) monomer yield was two to three times (0.48 to 0.73 mol of 3HV mol of propionate consumed(sup-1)) that observed in a control experiment (0.25 mol mol(sup-1)), where the accumulation-phase dissolved-oxygen concentration was 50 to 70% of air saturation. The overall polymer productivity of the fermentation was somewhat decreased by low dissolved-oxygen contents, owing to a slower 3HB production rate. The effect of a low dissolved-oxygen concentration is probably attributable to a reduction of the oxygen-requiring decarbonylation of propionyl-coenzyme A (CoA) to acetyl-CoA.     

Interaction between the formyl group of heme a and arginine 54 in cytochrome aa(3) from Paracoccus denitrificans

The optical spectrum of heme a is red-shifted in aa(3)-type cytochrome c oxidases compared to isolated low-spin heme A model compounds. Early spectroscopic studies indicated that this may be due to hydrogen-bonding of the formyl group of heme a to an amino acid in the close vicinity. Here we show that most of the optical spectral shift of native heme a is due to a hydrogen-bonding interaction between the formyl group and arginine-54 in subunit I of cytochrome aa(3) from Paracoccus denitrificans, and that a smaller part is due to an electrostatic interaction between the D ring propionate of heme a and arginine-474.     

Kinetics of iron acquisition from ferric siderophores by Paracoccus denitrificans.

The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to 55Fe-labeled ferric siderophore chelates is described. Iron is acquired from the ferric chelate of the natural siderophore L-parabactin in a process exhibiting biphastic kinetics by Lineweaver-Burk analysis. The kinetic data for 1 microM less than [Fe L-parabactin] less than 10 microM fit a regression line which suggests a low-affinity system (Km = 3.9 +/- 1.2 microM, Vmax = 494 pg-atoms of 55Fe min-1 mg of protein-1), whereas the data for 0.1 microM less than or equal to [Fe L-parabactin] less than or equal to 1 microM fit another line consistent with a high-affinity system (Km = 0.24 +/- 0.06 microM, Vmax = 108 pg-atoms of 55Fe min-1 mg of protein-1). The Km of the high-affinity uptake is comparable to the binding affinity we had previously reported for the purified ferric L-parabactin receptor protein in the outer membrane. In marked contrast, ferric D-parabactin data fit a single regression line corresponding to a simple Michaelis-Menten process with comparatively low affinity (Km = 3.1 +/- 0.9 microM, Vmax = 125 pg-atoms of 55Fe min-1 mg of protein-1). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-homoparabactin, L-agrobactin, and L-vibriobactin) also exhibit biphasic kinetics with a high-affinity component similar to ferric L-parabactin. Circular dichroism confirmed that these ferric chelates, like ferric L-parabactin, exist as the lambda enantiomers. The A forms ferric parabactin (ferrin D- and L-parabactin A), in which the oxazoline ring is hydrolyzed to the open-chain threonyl structure, exhibit linear kinetics with a comparatively high Km (1.4 +/- 0.3 microM) and high Vmax (324 pg-atoms of 55Fe min-1 of protein-1). Furthermore, the marked stereospecificity seen between ferric D- and L-parabactins is absent; i.e., iron acquisition from ferric parabactin A is non stereospecific. The mechanistic implications of these findings in relation to a stereospecific high-affinity binding followed by a nonstereospecific postreceptor processing is discussed.     

Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation

An efficient method for the transformation of the methylotrophic bacterium Methylobacterium extorquens NR-2 with a broad-host-range plasmid, pLA2917, by electroporation was examined. Transformants of M. extorquens NR-2 expressing resistance to kanamycin were obtained after electric pulse. These transformants were found to harbor a single plasmid which was electrophoretically identical and homologous to pLA2917 obtained from Escherichia coli. Several factors which determined the transformation efficiency were optimized, resulting in a transformation efficiency of up to 8 × 10³ transformants per μg of plasmid DNA by 10 pulses at a field strength of 10 kV/cm and a pulse duration of 300 μs.     

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

Purification,characterization and crystallization of the F-ATPase from Paracoccus denitrificans

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.