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Characterizing proteins in action requires appropriate biophysical techniques sensitive to protein motions. One of the technique dedicated to monitor protein dynamics is Site-Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR). The main purpose of this chapter is to describe and illustrate the different strategies based on the use of nitroxide spin labels either as reporters or as a means to measure inter-label distances. The complementarity of these different approaches to answer biological questions will be addressed. The objective is also to give non-specialist readers an overview of the recent developments in the field of SDSL-EPR dedicated to the study of protein dynamics. A particular emphasis will be devoted to describe the design and application of new nitroxide spin labels that allow overcoming the limitations of the classical ones.

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Molybdoenzymes are ubiquitous and play important roles in all kingdoms of life. The cofactors of these enzymes comprise the metal, molybdenum (Mo), which is bound to a special organic ligand system called molybdopterin (MPT). Additional small ligands are present at the Mo atom, including water, hydroxide, oxo-, sulfido-, or selenido-functionalities, and in some enzymes, amino acid ligand, such as serine, aspartate, cysteine, or selenocysteine that coordinate the cofactor to the peptide chain of the enzyme. The so-called molybdenum cofactor (Moco) is deeply buried within the protein at the end of a narrow funnel, giving access only to the substrate. In 1974, an assay was developed by Nason and coworkers using the pleiotropic Neurospora crassa mutant, nit-1, for the reconstitution of molybdoenzyme activities from crude extracts. These studies have led to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since then by using specific molybdenum enzymes as the source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and subsequently inserted into apo-molybdoenzymes with the assistance of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli trimethylamine N-oxide (TMAO) reductase.

International audience; Hydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H2 metabolism.

Biofilm formation is a complex process resulting from the action of imbricated pathways in response to environmental cues. In this study, we showed that biofilm biogenesis in the opportunistic pathogen Pseudomonas aeruginosa depends on the availability of RpoS, the sigma factor regulating the general stress response in bacteria. Moreover, it was demonstrated that RpoS is post-translationally regulated by the HsbR-HsbA partner switching system as has been demonstrated for its CrsR-CrsA homolog in Shewanella oneidensis. Finally, it was established that HsbA, the anti-sigma factor antagonist, has a pivotal role depending on its phosphorylation state since it binds HsbR, the response regulator, when phosphorylated and FlgM, the anti-sigma factor of FliA, when non-phosphorylated. The phosphorylation state of HsbA thus drives the switch between the sessile and planktonic way of life of P. aeruginosa by driving the release or the sequestration of one or the other of these two sigma factors.

International audience; Aquatic plants, comprising different divisions of embryophytes, derive from terrestrial ancestors. They have evolved to live in water, both fresh and salty, an environment that presents unique challenges and opportunities for photosynthesis and growth. These include, compared to air, a low water stress, a greater density, and attenuation of light, and a more variable supply of inorganic carbon, both in concentration and chemical species, but overall a lower carbon availability, and the opportunity to take up nutrients from the water. The leaves of many aquatic plants are linear, dissected, whorled, or cylindrical with a large volume of air spaces. They tend to have a high specific leaf area, thin cuticles, and usually lack functional stomata. Exploiting the availability of chemicals in their environment, freshwater macrophytes may incorporate silica in their cell wall, while seagrasses contain sulphated polysaccharides, similar to those of marine macroalgae; both groups have low lignin content. This altered cell wall composition produces plants that are more flexible and therefore more resistant to hydraulic forces (mechanical stress arising from water movement). Aquatic plants may have enhanced light harvesting complexes conferring shade adaptation, but also have mechanisms to cope with high light. Aquatic plants have evolved numerous strategies to overcome potential carbon-limitation in water. These include growing in micro-environments where CO2 is high, producing leaves and roots that exploit CO2 from the air or sediment and operating concentrating mechanisms that increase CO2 (CCM) around the primary carboxylating enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. These comprise C4 metabolism, crassulacean acid metabolism, and the ability to exploit the often high concentrations of HCO3−, and ~50% of freshwater macrophytes and ~85% of seagrasses have one or more CCM. Many of these adaptations involve trade-offs between conflicting constraints and opportunities while others represent ‘synergies’ that help to maximize the productivity of this important group of plants.

International audience; Hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus was immobilized in MgO-templated carbon (MgOC). Two different pore sizes were investigated, large pore size of 150 nm (MgOC150) and smaller pore size of 35 nm (MgOC35). Direct H2 oxidation proceeded in both MgOC150 and MgOC35. Hydrogenase embedded in the carbon material exhibited the expected properties in terms of onset for H2 oxidation and kinetics of formation of the inactive state at high potentials, whatever the size of the pores. Pore size much larger than the size of the enzyme favored the loading of the enzyme, yielding to high catalytic current reported to the capacitance. Pore size closer to the enzyme diameter, as determined by DLS, enhanced the stability of the enzyme at high temperature.

International audience; Scavenger receptors (SRs) like cluster determinant 36 (CD36) and SR class B type I (SR-BI) play a debated role in lipid transport across the intestinal brush border membrane. We used surface plasmon resonance to analyze real-time interactions between the extracellular protein loops and various ligands ranging from single lipid molecules to mixed micelles. Micelles mimicking physiological structures were necessary for optimal binding to both the extracellular loop of CD36 (lCD36) and the extracellular loop of SR-BI (lSR-BI). Cholesterol, phospholipid, and fatty acid micellar content significantly modulated micelle binding to and dissociation from the transporters. In particular, high phospholipid micellar concentrations inhibited micelle binding to both receptors (−53.8 and −74.4% binding at 0.32 mM compared with 0.04 mM for lCD36 and lSR-BI, respectively, P < 0.05). The presence of fatty acids was crucial for micelle interactions with both proteins (94.4 and 81.3% binding with oleic acid for lCD36 and lSR-BI, respectively, P < 0.05) and fatty acid type substitution within the micelles was the component that most impacted micelle binding to the transporters. These effects were partly due to subsequent modifications in micellar size and surface electric charge, and could be correlated to micellar vitamin D uptake by Caco-2 cells. Our findings show for the first time that micellar lipid composition and micellar properties are key factors governing micelle interactions with SRs.

International audience; The kinetics and isotherms of adsorption of Myrothecium verrucaria bilirubin oxidase (Mv BOD) on nanoporous carbons (CNP) and carbon nanofibers (CNF) were studied by the solution depletion method. The kinetics of adsorption of Mv BOD on both carbons are very fast, reaching equilibrium within 10 min and 30 min for CNP and CNF, respectively. The adsorption isotherms reveal a strong affinity between Mv BOD and both carbons. An original flow-through device based on electrodes filled with CNP and CNF connected to an UV–vis spectrophotometer was used to correlate the dioxygen catalytic current reduction to the amount of Mv BOD adsorbed on the carbons. It was shown that although the amount of BOD adsorbed on CNP is much lower than the amount adsorbed on CNF, the currents are comparable, suggesting that the Mv BOD orientation is more favourable for direct electron transfer in the case of CNP. Chronoamperometry experiments showed that the catalytic current is stable for a few days (70 h). It was emphasized by stop and flow experiments that the current is limited by mass transport inside the column.

The concentration of CO2 in many aquatic systems is variable, often lower than the KM of the primary carboxylating enzyme Rubisco, and in order to photosynthesize efficiently, many algae operate a facultative CO2 concentrating mechanism (CCM). Here we measured the responses of a marine diatom, Thalassiosira pseudonana, to high and low concentrations of CO2 at the level of transcripts, proteins and enzyme activity. Low CO2 caused many metabolic pathways to be remodeled. Carbon acquisition enzymes, primarily carbonic anhydrase, stress, degradation and signaling proteins were more abundant while proteins associated with nitrogen metabolism, energy production and chaperones were less abundant. A protein with similarities to the Ca2+/ calmodulin dependent protein kinase II_association domain, having a chloroplast targeting sequence, was only present at low CO2. This protein might be a specific response to CO2 limitation since a previous study showed that other stresses caused its reduction. The protein sequence was found in other marine diatoms and may play an important role in their response to low CO2 concentration.

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) both free and in complex with the X domain (XD, aa 459-507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N(TAIL) variants, we showed that the 490-500 region of N(TAIL) is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the alpha-helical sampling of the free form. In addition, we showed that the 505-525 region of N(TAIL) conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N(TAIL)-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488-525 region of N(TAIL), in particular the unfolding of the alpha-helical region when the temperature was increased from 281 K to 310 K.

More than a decade ago, Heliobacteria were recognised to contain a Rieske/cytb complex in which the cytochrome b subunit is split into two separate proteins, a peculiar feature characteristic of the cyanobacterial and plastidic b 6 f complex. The common presence of RCI-type reaction centres further emphasise possible evolutionary links between Heliobacteria, Chlorobiaceae and Cyanobacteria. In this contribution, we further explore the evolutionary relationships among these three phototrophic lineages by both molecular phylogeny and consideration of phylogenetic marker traits of the superfamily of Rieske/cytb complexes. The combination of these two methods suggests the existence of a “green” clade involving many non-phototrophs in addition to the mentioned RCI-type photosynthetic organisms. Structural and functional idiosyncrasies are (re-)interpreted in the framework of evolutionary biology and more specifically evolutionary bioenergetics.

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood.

CP12 is a flexible protein that is well-known to interact with GAPDH, and this association is crucial to the regulation of enzyme activity. This regulation is likely related to structural transitions of both proteins, but the molecular bases of these changes are not yet understood. To answer this issue, we undertook a study based on the use of paramagnetic probes grafted on cysteine residues and followed by EPR spectroscopy. We present a new application of this approach that enables us to probe the functional role of cysteine residues in protein-protein interactions. Algal CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state and has some alpha-helical secondary structural elements. In contrast, in its reduced state, CP12 is mainly unstructured and shares some physical properties with intrinsically disordered proteins. Treatment of CP12 with a methane thiosulfonate derivative spin-label (MTSL) led to the labeling of the cysteine residues involved in the C-terminal bridge only as revealed by mass spectrometry. Surprisingly, the partner protein GAPDH induced the cleavage of the disulfide bridge between the cysteine residues of CP12 and the spin-label, resulting in the full release of the label. We showed the existence of a transitory interaction between both proteins and proposed a mechanism based on a thiol-disulfide exchange reaction. The results of this study point out a novel role of the algal GAPDH which is often termed a "moonlighting" protein.

Evolutionary histories of enzymes involved in chemiosmotic energy conversion indicate that a strongly oxidizing substrate was available to the last universal common ancestor before the divergence of Bacteria and Archaea. According to palaeogeochemical evidence, O(2) was not present beyond trace amounts on the early Earth. Based on recent phylogenetic, enzymatic and geochemical results, we propose that, in the earliest Archaean, nitric oxide (NO) and its derivatives nitrate and nitrite served as strongly oxidizing substrates driving the evolution of a bioenergetic pathway related to modern dissimilatory denitrification. Aerobic respiration emerged later from within this ancestral pathway via adaptation of the enzyme NO reductase to its new substrate, dioxygen.

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International audience; Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane β barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner β-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the β barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.

International audience; The chloroplast protein CP12 is known to play a leading role in a complex formation with the enzymes GAPDH and PRK. As a preliminary step towards the understanding of the complex formation mechanism and the exact role of this protein linker, a comparative modelling of the CP12 protein of the green alga Chlamydomonas reinhardtii was performed. Because of the very few structural information and poor template similarities, the derivation of the model consisted in an iterative trial-and-error procedure using the comparative modelling program MODELLER, the following three structure validation programs PROCHECK, PROSA, and WHATIF, and molecular mechanics energy refinement of the model using the program CHARMM. The analysis of the final model reveals a scaffold of key residues that is believed to be essential in the folding mechanism and that coincides with the residues conserved throughout the CP12 family. Our results suggest that this protein is a typical disordered protein. Finally, the various mechanisms by which the CP12 protein can self-interact or binds to other enzymes are discussed in light of its modelled structure and characteristics.

Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

International audience; Le fonctionnement des enzymes qui catalysent des réactions redox fait intervenir des étapes très diverses (diffusion du substrat à l’intérieur de l’enzyme, réactions chimiques au site actif, transferts à longue distance d’électrons et de protons) et qui impliquent des sites de la protéine distants les uns des autres.Cet article illustre, en prenant pour exemple les enzymes qui catalysent l’oxydation réversible du dihydrogène, comment l’électrochimie peut maintenant être utilisée en combinaison avec d’autres approches comme la chimie théorique et la mutagenèse dirigée, pour étudier des aspects variés du mécanisme moléculaire des enzymes redox.

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The work presented by Matsumura et al. in this issue of Structure describes the structure of the ternary GAPDH-NAD-CP12 and the binary NAD-GAPDH complex in the cyanobacterium Synechococcus elongatus.

Electrochemistry of polyheme bacterial cytochrome c 3 and catalytic oxidation of hydrogen by two different bacterial [NiFe] hydrogenases were investigated for the first time in pure room-temperature ionic liquids (RTILs) as electrolyte. Direct electrochemical response of Desulfovibrio vulgaris Hildenborough cytochrome c 3 (DvH cytc 3 ) adsorbed at a pyrolytic graphite (PG) electrode was observed in the RTILs used in this work: 1-butyl-3-methylimidazolium tetrafluoroborate (BmimBF4), 1-ethyl-3-methylimidazolium tetrafluoroborate (EmimBF4) and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (EmimNTf2). The electrochemical signal differed however from that obtained in aqueous buffer, and depended on the type of RTIL. UV–vis measurements as well as transfer experiments from aqueous buffer to RTILs or RTILs to aqueous buffer strongly suggested that the protein was not denatured in the presence of RTILs. EmimNTf2, as a hydrophobic non-water-miscible RTIL, was demonstrated to stabilize the native form of DvH cytc 3 . Moreover it allowed an amount of electroactive DvH cytc 3 30-fold higher than observed in aqueous buffer. Catalytic oxidation of H 2 via Desulfovibrio fructosovorans [NiFe] hydrogenase (Df Hase) mediated by DvH cytc 3 failed however. Further investigation suggested that Df Hase could be inhibited in the presence of RTILs. Reasons for such an inhibition were explored, including the blocking up of the substrate channels. By using hyperthermophilic [NiFe] membrane-bound hydrogenase from Aquifex aeolicus (Aa Hase) an efficient direct catalytic oxidation process was obtained in mixed aqueous buffer/RTILs electrolytes, although direct H 2 oxidation was not observed in pure RTIL. Chronoamperometric experiments showed that Aa Hase could afford 80% RTILs in aqueous buffer, thus giving the opportunity of future electrolytes with uncommon and variable properties for biofuel cell design.

The nature of life has been a topic of interest from the earliest of times, and efforts to explain it in mechanistic terms date at least from the 18th century. However, the impressive development of molecular biology since the 1950s has tended to have the question put on one side while biologists explore mechanisms in greater and greater detail, with the result that studies of life as such have been confined to a rather small group of researchers who have ignored one another's work almost completely, often using quite different terminology to present very similar ideas. Central among these ideas is that of closure, which implies that all of the catalysts needed for an organism to stay alive must be produced by the organism itself, relying on nothing apart from food (and hence chemical energy) from outside. The theories that embody this idea to a greater or less degree are known by a variety of names, including (M,R) systems, autopoiesis, the chemoton, the hypercycle, symbiosis, autocatalytic sets, sysers and RAF sets. These are not all the same, but they are not completely different either, and in this review we examine their similarities and differences, with the aim of working towards the formulation of a unified theory of life.

Such a difficulty was recently encountered in the study of a small and flexible chloroplast protein CP12 from the green alga Chlamydomonas reinhardtii by spin-labeling EPR spectroscopy. In this organism, CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state. Although the introduction of spin labels at the two cysteine residues of the C-terminal disulfide bridge enabled us to identify a new role of the partner protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH), it precluded the direct study of the complex formation GAPDH/CP12. [4] To overcome this difficulty, grafting of the nitroxide probe to residues other than cysteines is required. One strategy using a genetically encoded unnatural amino acid has been recently proposed. [5] The incorporation of such unnatural amino acids relies, however, on a rather complex strategy involving an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid. As such a strategy is difficult to set up, we propose an alternative method consisting of selectively targeting residues other than cysteines with a nitroxide probe. Bioconjugation of small molecules to protein residues is very challenging, and several reactions have recently been proposed to target specific residues selectively. [6] In particular, efforts have been paid to modify the aromatic amino acid side chains of tryptophan [7] and tyrosine. [8] Among them, a threecomponent Mannich-type reaction has been developed that allows the modification of tyrosine under mild, biocompatible, and metal-free conditions. [8a] Inspired by these recent studies, we present the selective grafting of a nitroxide probe to tyrosine by using the Mannich-type reaction on CP12, a protein bearing only one natural tyrosine residue. This unique tyrosine residue, located at position 78 in the sequence of a total of 80 amino acids, makes this protein an ideal candidate for demonstrating the feasibility of tyrosine-targeted spin

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Overexpression of a protein in a foreign host is often the only route toward an exhaustive characterization, especially when purification from the natural source(s) is hardly achievable. The key issue in these studies relies on quality control of the purified recombinant protein to precisely determining its identity as well as any undesirable microheterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top-down tandem mass spectrometry (MSMS) requires the use of highly accurate and highly resolved experiments to reveal subtle sequence modifications. In the present study, the top-down MSMS approach combined with traveling wave ion mobility (TWIM) separation was evaluated for its ability to achieve high sequence coverage and to reveal subtle microheterogeneities that were hitherto only accessible with Fourier-transform ion cyclotron resonance-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both the wild type and K496C variant of the recombinant X domain (XD; aa's 459-507) of the measles virus phosphoprotein expressed in Escherichia coli . Using top-down MSMS combined with TWIM, we show that XD samples occasionally exhibit a microheterogeneity that could not be anticipated from the nucleotide sequence of the encoding constructs and that likely reflects a genetic drift, neutral or not, occurring during expression. In addition, a 1-oxyl-2,2,5,5-tetramethyl-δ3-pyrroline-3-methyl methanethiosulfonate nitroxide probe that was grafted onto the K496C XD variant was shown to undergo oxidation and/or protonation in the electrospray ionization source, leading to artifactual mass increases.

Wolfe-Simon et al . (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) argued that the bacterial strain GFAJ-1 can vary the elemental composition of its biomolecules by substituting arsenic for phosphorus. Although their data show that GFAJ-1 is an extraordinary extremophile, consideration of arsenate redox chemistry undermines the suggestion that arsenate can replace the physiologic functions of phosphate.

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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.

Chemical equations are normally written in terms of specific ionic and elemental species and balance atoms of elements and electric charge. However, in a biochemical context it is usually better to write them with ionic reactants expressed as totals of species in equilibrium with each other. This implies that atoms of elements assumed to be at fixed concentrations, such as hydrogen at a specified pH, should not be balanced in a biochemical equation used for thermodynamic analysis. However, both kinds of equations are needed in biochemistry. The apparent equilibrium constant K' for a biochemical reaction is written in terms of such sums of species and can be used to calculate standard transformed Gibbs energies of reaction Δ(r)G'°. This property for a biochemical reaction can be calculated from the standard transformed Gibbs energies of formation Δ(f)G(i)'° of reactants, which can be calculated from the standard Gibbs energies of formation of species Δ(f)G(j)° and measured apparent equilibrium constants of enzyme-catalyzed reactions. Tables of Δ(r)G'° of reactions and Δ(f)G(i)'° of reactants as functions of pH and temperature are available on the web, as are functions for calculating these properties. Biochemical thermodynamics is also important in enzyme kinetics because apparent equilibrium constant K' can be calculated from experimentally determined kinetic parameters when initial velocities have been determined for both forward and reverse reactions. Specific recommendations are made for reporting experimental results in the literature.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. The Cytochrome bo3 ubiquinol oxidase (QOX) from Escherichia coli (E. coli) contains a redox-active quinone, the so-called “high-affinity” QH quinone. The location of this cofactor and its binding site has yet to be accurately determined by X-ray crystallographic studies. Based on site-directed mutagenesis studies, a putative quinone binding site in the protein has been proposed. The exact binding partner of this cofactor and also whether it is stabilised as an anionic semiquinone or as a neutral radical species is a matter of some speculation. Both Hyperfine Sub-level Correlation (HYSCORE) and Double Nuclear Coherence Transfer Spectroscopy (DONUT-HYSCORE) spectroscopy as well as density functional theory (DFT) have been applied to investigate the QH binding site in detail to resolve these issues. Use is made of site-directed variants as well as globally 15N/14N-exchanged protein. Comparison of computed and experimental 13C hyperfine tensors provides strong support for the binding of the semiquinone radical in an anionic rather than a neutral protonated form. These results are compared with the corresponding information available on other protein binding sites and/or on model systems and are discussed with regard to the location and potential function of QH in the overall mechanism of function of this family of haem copper oxidases.

The [NiFe] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic Aquifex aeolicus bacterium (Aa Hase) presents oxygen, carbon monoxide, and temperature resistances. Since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. Efficient immobilization of the enzyme onto electrodes is however a mandatory step. To gain further insight into the parameters governing the interfacial electron process, cyclic voltammetry was performed combining the use of a phenothiazine dye with a membrane electrode design where the enzyme is entrapped in a thin layer. In the absence of the phenothiazine dye, direct electron transfer (DET) for H(2) oxidation is observed due to Aa Hase adsorbed onto the PG electrode. An unexpected loss of the catalytic current with time is however observed. The effect of toluidine blue O (TBO) on the catalytic process is first studied with TBO in solution. In addition to the expected mediated electron transfer process (MET), TBO is demonstrated to reconnect directly some Aa Hase molecules possibly released from the electrode but still entrapped in the thin layer. On adsorbed TBO the two same processes occur demonstrating the ability of the TBO film to connect Aa Hase via a DET process. Loss of activity is however observed due to the poor stability of adsorbed TBO at high temperatures. Aa Hase immobilization is then studied on electropolymerized TBO (pTBO). The effect of film thickness, temperature, presence of inhibitors and pH is evaluated. Given a film thickness less than 20 nm, H(2) oxidation proceeds via a mixed DET/MET process through the pTBO film. A high and very stable H(2) oxidation activity is reached, showing the potential applicability of the bioelectrode for biotechnologies. Finally, the multifunctional roles of TBO-based matrix are underlined, including redox mediator, Aa Hase anchor, but also buffering and ROS scavenger capabilities to drive pH local changes and avoid oxidative damage.

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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 (H(2)S) to molecular oxygen. H(2)S 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 bc(1) complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c(555) and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba(3)-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O(2) in the presence of the electron donor, H(2)S. 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.

Hsp33, a molecular chaperone specifically activated by oxidative stress conditions that lead to protein unfolding, protects cells against oxidative protein aggregation. Stress sensing in Hsp33 occurs via its C-terminal redox switch domain, which consists of a zinc center that responds to the presence of oxidants and an adjacent metastable linker region, which responds to unfolding conditions. Here we show that single mutations in the N terminus of Hsp33 are sufficient to either partially (Hsp33-M172S) or completely (Hsp33-Y12E) abolish this post-translational regulation of Hsp33 chaperone function. Both mutations appear to work predominantly via the destabilization of the Hsp33 linker region without affecting zinc coordination, redox sensitivity, or substrate binding of Hsp33. We found that the M172S substitution causes moderate destabilization of the Hsp33 linker region, which seems sufficient to convert the redox-regulated Hsp33 into a temperature-controlled chaperone. The Y12E mutation leads to the constitutive unfolding of the Hsp33 linker region thereby turning Hsp33 into a constitutively active chaperone. These results demonstrate that the redox-controlled unfolding of the Hsp33 linker region plays the central role in the activation process of Hsp33. The zinc center of Hsp33 appears to act as the redox-sensitive toggle that adjusts the thermostability of the linker region to the cell redox status. In vivo studies confirmed that even mild overexpression of the Hsp33-Y12E mutant protein inhibits bacterial growth, providing important evidence that the tight functional regulation of Hsp33 chaperone activity plays a vital role in bacterial survival.

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The ctpA (ccoI) gene product, a putative inner membrane copper-translocating P1B-type ATPase present in many bacteria, has been shown to be involved only in the cbb(3) assembly in Rhodobacter capsulatus and Bradyrhizobium japonicum. ctpA was disrupted in Rubrivivax gelatinosus, and the mutants showed a drastic decrease in both cbb(3) and caa(3) oxidase activities. Inactivation of ctpA results also in a decrease in the amount of the nitrous oxide reductase, NosZ. This pleiotropic phenotype could be partially rescued by excess copper in the medium, indicating that CtpA is likely a copper transporter that supplies copper-requiring proteins in the membrane with this metal. Although CtpA shares significant sequence homologies with the homeostasis copper efflux P1B-type ATPases including the bacterial CopA and the human ATP7A and ATP7B, disruption of ctpA did not result in any sensitivity to excess copper. This indicates that the CtpA is not crucial for copper tolerance but is involved in the assembly of membrane and periplasmic copper enzymes in this bacterium. The potential roles of CtpA in bacteria in comparison with CopA are discussed.

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Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV), whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555, 54-59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392); this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the "solution potential" does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site.

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The [NiFe] hydrogenase (Hase I) involved in the aerobic respiration of the hyperthermophilic bacterium Aquifex aeolicus shows increased oxygen tolerance and thermostability and can form very stable films on pyrolytic graphite electrodes. Oxygen-tolerant enzymes, like the ones from A. aeolicus and Ralstonia eutropha, are reported to be insensitive to CO inhibition. This is in contrast to known and well-characterized (oxygen-sensitive) hydrogenases, for which carbon monoxide is a competitive inhibitor. In this study, the interaction of Hase I from A. aeolicus with CO is examined using in situ infrared electrochemistry and time-resolved FTIR spectroscopy. We could observe the formation of a CO adduct state, a finding that set the grounds to investigate the affinity of an O(2)-tolerant enzyme for binding CO as well as the reversibility of this process. In the case of A. aeolicus, this extrinsic CO is shown to be weakly attached and the adduct state is light-sensitive at low temperatures. The energetic parameters for the rebinding of CO at the active site were estimated from the rate constants of this process after photolysis and the results compared to those obtained for standard hydrogenases. Formation of a weak Ni-CO bond in the active site of Hase I most likely results from the different interaction of this enzyme with inhibitors and/or different active site electronic properties to which non standard amino acid residues in the vicinity of the active site might contribute.

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Data on structure and function of the Rieske/cytb complex from Heliobacteria are scarce. They indicate that the complex is related to the b (6) f complex in agreement with the phylogenetic position of the organism. It is composed of a diheme cytochrome c, and a Rieske iron-sulfur protein, together with transmembrane cytochrome b (6) and subunit IV. Additional small subunits may be part of the complex. The cofactor content comprises heme c (i), first discovered in the Q(i) binding pocket of b (6) f complexes. The redox midpoint potentials are more negative than in b (6) f complex in agreement with the lower redox midpoint potentials (by about 150 mV) of its reaction partners, menaquinone, and cytochrome c (553). The enzyme is implicated in cyclic electron transfer around the RCI. Functional studies are favored by the absence of antennae and the simple photosynthetic reaction chain but are hampered by the high oxygen sensitivity of the organism, its chlorophyll, and lipids.

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