Myriam Brugna

Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria

Abstract Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 μM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c3(Mr. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c3 from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c7 from Desulfuromonas acetoxidans. Results with cytochromes c3 and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate.

Daddy, where did (PS)I come from?

The reacton centre I (RCI)-type photosystems from plants, cyano-, helio- and green sulphur bacteria are compared and the essential properties of an archetypal RCI are deduced. Species containing RCI-type photosystems most probably cluster together on a common branch of the phylogenetic tree. The predicted branching order is green sulphur, helio- and cyanobacteria. Striking similarities between RCI- and RCII-type photosystems recently became apparent in the three-dimensional structures of photosystem I (PSI), PSII and RCII. The phylogenetic relationship between all presently known photosystems is analysed suggesting (a) RCI as the ancestral photosystem and (b) the descendence of PSII from RCI via gene duplication and gene splitting. An evolutionary model trying to rationalise available data is presented.

A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans.

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the CuA center of cytochrome c oxidase aa3. This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O2 reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD+(P) from ferrous iron (uphill pathway) required for CO2 fixation and other anabolic processes. Besides the proteins involved in the O2 reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc42), suggesting a possible physical link between these two pathways.

Original design of an oxygen-tolerant [NiFe] hydrogenase: major effect of a valine-to-cysteine mutation near the active site.

Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.

Biocatalysts for fuel cells: efficient hydrogenase orientation for H2 oxidation at electrodes modified with carbon nanotubes

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H2 oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H2 oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials.

A sequential electron transfer from hydrogenases to cytochromes in sulfate-reducing bacteria.

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26¿ omitted¿000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13¿ omitted¿000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13¿ omitted¿000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13¿ omitted¿000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.

The Qo-site inhibitor DBMIB favours the proximal position of the chloroplast Rieske protein and induces a pK-shift of the redox-linked proton

The interaction of the inhibitor 2,5‐dibromo‐3‐methyl‐6‐isopropylbenzoquinone (DBMIB) with the Rieske protein of the chloroplast b 6 f complex has been studied by EPR. All three redox states of DBMIB were found to interact with the iron‐sulphur cluster. The presence of the oxidised form of DBMIB altered the equilibrium distribution of the Rieske proteins conformational substates, strongly favouring the proximal position close to heme b L. In addition to this conformational effect, DBMIB shifted the pK‐value of the redox‐linked proton involved in the iron‐sulphur clusters redox transition by about 1.5 pH units towards more acidic values. The implications of these results with respect to the interaction of the native quinone substrate and the Rieske cluster in cytochrome bc complexes are discussed.

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. II. The Rieske protein of phylogenetically distant acidophilic organisms.

The Rieske proteins of two phylogenetically distant acidophilic organisms, i.e. the proteobacteriumThiobacillus ferrooxidans and the crenarchaeonSulfolobus acidocaldarius, were studied by EPR. Redox titrations at a range of pH values showed that the Rieske centers of both organisms are characterized by redox midpoint potential-versus-pH curves featuring a common pK value of 6.2. This pK value is significantly more acidic (by almost 2 pH units) than that of Rieske proteins in neutrophilic species. The orientations of the Rieske center’s g tensors with respect to the plane of the membrane were studied between pH 4 and 8 using partially ordered samples. At pH 4, theSulfolobus Rieske cluster was found in the “typical” orientation of chemically reduced Rieske centers, whereas this orientation changed significantly on going toward high pH values. TheThiobacillus protein, by contrast, appeared to be in the “standard” orientation at both low and high pH values. The results are discussed with respect to the molecular parameters conveying acid resistance and in light of the recently demonstrated long-range conformational movement of the Rieske protein during enzyme turnover in cytochrome bc 1 complexes.

New Functional Sulfide Oxidase-Oxygen Reductase Supercomplex in the Membrane of the Hyperthermophilic Bacterium Aquifex aeolicus

Aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. It was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (H2S) to molecular oxygen. H2S is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. We have purified and characterized a novel membrane-bound multienzyme supercomplex that brings together all the molecular components involved in this bioenergetic chain. Our results indicate that this purified structure consists of one dimeric bc1 complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c555 and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba3-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O2 in the presence of the electron donor, H2S. This is the first demonstration of the existence of such a respirasome carrying a sulfide oxidase-oxygen reductase activity. Moreover, the kinetic properties of the sulfide quinone reductase change slightly when integrated in the supercomplex, compared with the free enzyme. We previously purified a complete respirasome involved in hydrogen oxidation and sulfur reduction from Aquifex aeolicus. Thus, two different bioenergetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. A model for the energetic sulfur metabolism of Aquifex aeolicus is proposed.

The membrane-extrinsic domain of cytochrome b558/566 from the Archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface

The orientation of the membrane‐attached cytochrome b 558/566‐haem with respect to the membrane was determined by electron paramagnetic resonance spectroscopy on two‐dimensionally ordered oxidised membrane fragments from Sulfolobus acidocaldarius. Unlike the other redox centres in the membrane, the cytochrome b 558/566‐haem was found to cover a range of orientations between 25° and 90°. The described results are reminiscent of those obtained on the Rieske cluster of bc complexes and indicate that the membrane‐extrinsic domain of cytochrome b 558/566 can perform pivoting motion between two extreme positions. Such a conformational flexibility is likely to play a role in electron transfer with its redox partners.