Laetitia Pieulle

Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate

Oxidative decarboxylation of pyruvate to form acetyl–coenzyme A, a crucial step in many metabolic pathways, is carried out in most aerobic organisms by the multienzyme complex pyruvate dehydrogenase. In most anaerobes, the same reaction is usually catalyzed by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFOR). Thus, PFOR is a potential target for drug design against certain anaerobic pathogens. Here, we report the crystal structures of the homodimeric Desulfovibrio africanus PFOR (data to 2.3 Å resolution), and of its complex with pyruvate (3.0 Å resolution). The structures show that each subunit consists of seven domains, one of which affords protection against oxygen. The thiamin pyrophosphate (TPP) cofactor and the three [4Fe–4S] clusters are suitably arranged to provide a plausible electron transfer pathway. In addition, the PFOR–pyruvate complex structure shows the noncovalent fixation of the substrate before the catalytic reaction.

Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus

We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.

Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase

The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.

Biochemical studies of the c-type cytochromes of the sulfate reducer Desulfovibrio africanus. Characterization of two tetraheme cytochromes c3 with different specificity

Three c-type cytochromes were isolated and characterized from the sulfate reducer Desulfovibrio africanus. A basic tetraheme cytochrome c3 of molecular mass 16 kDa was previously described and we have extended its characterization. Two other c3-type cytochromes, not previously observed, have also been characterized. These include an acidic tetraheme cytochrome c3 of molecular mass 15 kDa and an octaheme dimeric cytochrome c3 with a native size of 35 kDa. This is the first report of the presence of two distinct tetraheme cytochromes c3 in a Desulfovibrio species. The physico-chemical properties of the three cytochromes, including optical properties, iron content, cysteine and histidine content, N-terminal amino sequence and redox properties, are characteristic of cytochrome c3 family. The acidic tetraheme cytochrome c3 exhibited similar midpoint potential values for all four hemes (Em1 = -210 mV; Em2 = -240 mV; Em3 = -260 mV; Em4 = -270 mV), whereas in the basic tetraheme cytochrome c3 one heme had a much more positive potential than the others (Em1 = -90 mV; Em2 = -260 mV; Em3 = -280 mV; Em4 = -290 mV). The acidic tetraheme cytochrome c3 exhibited unique properties including amino-acid composition and poor reactivity towards hydrogenase. However, it is readily reduced by this enzyme in the presence of the basic cytochrome c3. The weak reactivity of the acidic tetraheme cytochrome c3 towards hydrogenase has been correlated with its low content of basic residues.

Further characterization of the two tetraheme cytochromes c3 from Desulfovibrio africanus: nucleotide sequences, EPR spectroscopy and biological activity

The genes encoding the basic and acidic tetraheme cytochromes c3 from Desulfovibrio africanus have been sequenced. The corresponding amino acid sequences of the basic and acidic cytochromes c3 indicate that the mature proteins consist of a single polypeptide chain of 117 and 103 residues, respectively. Their molecular masses, 15102 and 13742 Da, respectively, determined by mass spectrometry, are in perfect agreement with those calculated from their amino acid sequences. Both D. africanus cytochromes c3 are synthesized as precursor proteins with signal peptides of 23 and 24 residues for the basic and acidic cytochromes, respectively. These cytochromes c3 exhibit the main structural features of the cytochrome c3 family and contain the 16 strictly conserved cysteine + histidine residues directly involved in the heme binding sites. The D. africanus acidic cytochrome c3 differs from all the other homologous cytochromes by its low content of basic residues and its distribution of charged residues in the amino acid sequence. The presence of four hemes per molecule was confirmed by EPR spectroscopy in both cytochromes c3. The g-value analysis suggests that in both cytochromes, the angle between imidazole planes of the axial histidine ligands is close to 90 degrees for one heme and much lower for the three others. Moreover, an unusually high exchange interaction (approximately 10[-2] cm[-1]) was evidenced between the highest potential heme (-90 mV) and one of the low potential hemes in the basic cytochrome c3. The reactivity of D. africanus cytochromes c3 with heterologous [NiFe] and [Fe] hydrogenases was investigated. Only the basic one interacts with the two types of hydrogenase to achieve efficient electron transfer, whereas the acidic cytochrome c3 exchanges electrons specifically with the basic cytochrome c3. The difference in the specificity of the two D. africanus cytochromes c3 has been correlated with their highly different content of basic and acidic residues.

Oxygen reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases

Although Desulfovibrio vulgaris Hildenborough (DvH) is a strictly anaerobic bacterium, it is able to consume oxygen in different cellular compartments, including extensive periplasmic O₂ reduction with hydrogen as electron donor. The genome of DvH revealed the presence of cydAB and cox genes, encoding a quinol oxidase bd and a cytochrome c oxidase, respectively. In the membranes of DvH, we detected both quinol oxygen reductase [inhibited by heptyl-hydroxyquinoline-N-oxide (HQNO)] and cytochrome c oxidase activities. Spectral and HPLC data for the membrane fraction revealed the presence of o-, b- and d-type haems, in addition to a majority of c-type haems, but no a-type haem, in agreement with carbon monoxide-binding analysis. The cytochrome c oxidase is thus of the cc(o/b)o₃ type, a type not previously described. The monohaem cytochrome c₅₅₃ is an electron donor to the cytochrome c oxidase; its encoding gene is located upstream of the cox operon and is 50-fold more transcribed than coxI encoding the cytochrome c oxidase subunit I. Even when DvH is grown under anaerobic conditions in lactate/sulfate medium, the two terminal oxidase-encoding genes are expressed. Furthermore, the quinol oxidase bd-encoding genes are more highly expressed than the cox genes. The cox operon exhibits an atypical genomic organization, with the gene coxII located downstream of coxIV. The occurrence of these membrane-bound oxygen reductases in other strictly anaerobic Deltaproteobacteria is discussed.

An oxygen reduction chain in the hyperthermophilic anaerobe Thermotoga maritima highlights horizontal gene transfer between Thermococcales and Thermotogales.

The hyperthermophile Thermotoga maritima, although strictly anaerobic, is able to grow in the presence of low amounts of O(2). Here, we show that this bacterium consumes O(2) via a three-partner chain involving an NADH oxidoreductase (NRO), a rubredoxin (Rd) and a flavo-diiron protein (FprA) (locus tags: TM_0754, TM_0659 and TM_0755, respectively). In vitro experiments showed that the NADH-dependent O(2) consumption rate was 881.9 (± 106.7) mol O(2) consumed min(-1) per mol of FprA at 37°C and that water was the main end-product of the reaction. We propose that this O(2) reduction chain plays a central role in the O(2) tolerance of T. maritima. Phylogenetic analyses suggest that the genes coding for these three components were acquired by an ancestor of Thermotogales from an ancestor of Thermococcales via a single gene transfer. This event likely also involved two ROS scavenging enzymes (neelaredoxin and rubrerythrin) that are encoded by genes clustered with those coding for FprA, NRO and Rd in the ancestor of Thermococcales. Such genomic organization would have provided the ancestor of Thermotogales with a complete set of enzymes dedicated to O(2)-toxicity defence. Beside Thermotogales and Thermococcales, horizontal gene transfers have played a major role in disseminating these enzymes within the hyperthermophilic anaerobic prokaryotic communities, allowing them to cope with fluctuating oxidative conditions that exist in situ.

Study of the Thiol/Disulfide Redox Systems of the Anaerobe Desulfovibrio vulgaris Points Out Pyruvate:Ferredoxin Oxidoreductase as a New Target for Thioredoxin 1

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent Km value for Trx1 of 8.9 μm. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (Km for NADPH, 743 μm; Km for NADH, 5.6 μm), and Trx3 was unable to reduce insulin. The Km value of TR3 for Trx3 was 1.12 μm. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1C33S as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

Crystallization and preliminary crystallographic analysis of the pyruvate–ferredoxin oxidoreductase from Desulfovibrio africanus

For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus. Two different orthorhombic (P212121 ) crystal forms have been grown with unit-cell dimensions a = 86.1, b = 146.7, c = 212.5 A and a = 84.8, b = 144.9, c = 203.0 A. Both crystals diffract to 2.3 A resolution using synchrotron radiation.