Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.     

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough

Abstract Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83 500 Da) is proposed to contain a molybdenum cofactor, a 27 000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14000 Da) holds a c -type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.     

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).     

Tungsten and molybdenum regulation of formate dehydrogenase expression in Desulfovibrio vulgaris Hildenborough

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC₃ protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC₃ specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.     

Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP complex--a membrane-bound redox complex involved in the sulfate respiratory pathway

Sulfate-reducing organisms use sulfate as an electron acceptor in an anaerobic respiratory process. Despite their ubiquitous occurrence, sulfate respiration is still poorly characterized. Genome analysis of sulfate-reducing organisms sequenced to date permitted the identification of only two strictly conserved membrane complexes. We report here the purification and characterization of one of these complexes, DsrMKJOP, from Desulfovibrio desulfuricans ATCC 27774. The complex has hemes of the c and b types and several iron-sulfur centers. The corresponding genes in the genome of Desulfovibrio vulgaris were analyzed. dsrM encodes an integral membrane cytochrome b; dsrK encodes a protein homologous to the HdrD subunit of heterodisulfide reductase; dsrJ encodes a triheme periplasmic cytochrome c; dsrO encodes a periplasmic FeS protein; and dsrM encodes another integral membrane protein. Sequence analysis and EPR studies indicate that DsrJ belongs to a novel family of multiheme cytochromes c and that its three hemes have different types of coordination, one bis-His, one His/Met, and the third a very unusual His/Cys coordination. The His/Cys-coordinated heme is only partially reduced by dithionite. About 40% of the hemes are reduced by menadiol, but no reduction is observed upon treatment with H2 and hydrogenase, irrespective of the presence of cytochrome c3. The aerobically isolated Dsr complex displays an EPR signal with similar characteristics to the catalytic [4Fe-4S]3+ species observed in heterodisulfide reductases. Further five different [4Fe-4S](2+/1+) centers are observed during a redox titration followed by EPR. The role of the DsrMKJOP complex in the sulfate respiratory chain of Desulfovibrio spp. is discussed.     

The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: purification and properties.

NAD-dependent formate dehydrogenase (FDH1) was isolated from the alpha-proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (alpha1beta1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme approximately 5 mol nonheme iron and acid-labile sulfur, 0.6 mol noncovalently bound FMN, and approximately 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the alpha-subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron-sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The beta-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron-sulfur cluster binding motif. The beta-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiquinone oxidoreductases.     

S-class cytochromes c have a variety of folding patterns: structure of cytochrome c-553 from Desulfovibrio vulgaris determined by the multi-wavelength anomalous dispersion method

The three-dimensional structure of cytochrome c-553 isolated from sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F strain, has been determined by the multi-wavelength anomalous dispersion technique with use of synchrotron radiation. The result shows that bacterial S-class cytochromes c have a variety of folding patterns. The relative location of two a-helices at amino- and carboxyl-terminals and the style of bonding to the heme group show "cytochrome c folding," but other regions of the structure are different from those of other cytochromes c previously reported. The results also give useful information about the location of sulfate-reducing bacterium on the phylogenetic tree of the bacterial cytochromes c superfamily.     

Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline.

The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.     

Identification of a fourth formate dehydrogenase in Methylobacterium extorquens AM1 and confirmation of the essential role of formate oxidation in methylotrophy

A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [¹³C]formate, as well as ¹⁴C-labeling experiments, demonstrated production of labeled CO₂ in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO₂ formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

Cytochrome spectrum of an obligate anaerobe, Eubacterium lentum.

An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air.     

Crystal structure of NAD-dependent formate dehydrogenase.

The ternary complex of NAD-dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 (enzyme-NAD-azide) has been crystallised in the space group P2(1)2(1)2(1) with cell dimensions a = 11.60 nm, b = 11.33 nm, c = 6.34 nm. There is 1 dimeric molecule/asymmetric unit. An electron density map was calculated using phases from multiple isomorphous replacement at 0.30 nm resolution. Four heavy atom derivatives were used. The map was improved by solvent flattening and molecular averaging. The atomic model, including 2 x 393 amino acid residues, was refined by the CORELS and PROLSQ packages using data between 1.0 nm and 0.30 nm excluding structure factors less than 1 sigma. The current R factor is 27.1% and the root mean square deviation from ideal bond lengths is 4.2 pm. The FDH subunit is folded into a globular two-domain (coenzyme and catalytic) structure and the active centre and NAD binding site are situated at the domain interface. The beta sheet in the FDH coenzyme binding domain contains an additional beta strand compared to other dehydrogenases. The difference in quaternary structure between FDH and the other dehydrogenases means that FDH constitutes a new subfamily of NAD-dependent dehydrogenases: namely the P-oriented dimer. The FDH nucleotide binding region of the structure is aligned with the three dimensional structures of four other dehydrogenases and the conserved residues are discussed. The amino acid residues which contribute to the active centre and which make contact with NAD have been identified.     

Highly purified hydroxylamine oxidoreductase derived from Nitrosomonas europaea. Some physicochemical and enzymatic properties.

Hydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied. The molecular weight of the enzyme as determined by gel filtration on Sephadex G150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175,000-180,000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17,500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (gamma peak), 521 (beta peak), 553 (alpha peak), and 460 nm (due to an unidentified chromophore). Although the alpha peak at 553 nm has a shoulder at 559 nm, the enzyme does not posses protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possess heme c molecules in different states. The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with "bacterial-type" cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. europaea) acts as an electron acceptor for the enzyme.     

The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II

The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.     

Purification and properties of cytochrome c-553, an electron acceptor for formate dehydrogenase of Desulfovibrio vulgaris, Miyazaki

Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the α-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is ?0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan.Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c₃, but is oxidized by ferricyanide or by O₂.Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H₂. The formate dehydrogenase does not reduce cytochrome c₃ in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.2.2.—.     

Coordination of the heme iron in the low-potential cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Different chirality of the axially bound methionine in the oxidized and reduced states

The coordination geometry at the heme iron of the cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans was investigated by 1H-nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with other c-type cytochromes. In contrast, a new structure was observed for the axial methionine in the reduced cytochromes c-553. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa and cytochrome c5 from Pseudomonas mendocina, which also contain S-chiral methionine, a different spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine prevails. For the ferricytochromes c-553 R chirality was found for the iron-bound sulfur. This is the first observation of different methionine chirality in different oxidation states of the same c-type cytochrome.     

甲醛氧化途径关键酶重组蛋白的生化特性及固定化酶吸收甲醛效果研究

甲醛脱氢酶(formaldehyde dehydrogenase,ADH)与甲酸脱氢酶(formate dehydrogenase,FDH)是甲醛氧化途径的两个关键酶.恶臭假单胞菌(Pseudomonas putida)的PADH是一种不依赖谷胱甘肽可以把游离甲醛直接氧化为甲酸的脱氢酶,博伊丁假丝酵母菌(Candida boidinii)的FDH在有NAD+存在时可以把甲酸氧化为二氧化碳.以基因组DNA为模板用PCR方法,从P.putida中扩增出PADH基因的编码区(padh),从C.boidinii中扩增出FDH的编码区(fdh),然后亚克隆到pET-28a(+)中分别构建这两个基因的原核表达载体pET-28a-padh和pET-28a-fdh,转化大肠杆菌,利用IPTG诱导重组蛋白PADH和FDH的表达.通过优化条件使重组蛋白的表达量占菌体总蛋白的70%以上,通过亲和层析法纯化出可溶性PADH和FDH重组蛋白.对重组蛋白的生化特性分析结果表明:PADH在最适反应温度50℃的活性为1.95 U/mg;FDH在最适反应温度40℃的活性为0.376 U/mg.所表达的重组蛋白与之前报道过的相比,具有更好的热稳定性和更广的温度适应范围.将PADH、FDH两个重组蛋白及辅因子NAD+固定到聚丙烯酰胺载体基质上,对固定化酶甲醛吸收效果的初步分析结果显示固定化酶对空气中的甲醛有一定的吸收效果,说明这两种酶被固定后具有开发成治理甲醛污染环保产品的潜力.