A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions.

Many bacteria of the genus are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments.

Allosteric Site on SHIP2 Identified Through Fluorescent Ligand Screening and Crystallography: A Potential New Target for Intervention.

Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P and PtdIns(3,4)P.

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels.

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches.

The Crystal Structure of a Biological Insulated Transmembrane Molecular Wire.

A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand β-barrel formed by MtrB.

Regioisomeric Family of Novel Fluorescent Substrates for SHIP2.

SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase, which, through its catalytic action on PtdInsP, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target, but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases.

An electrogenic redox loop in sulfate reduction reveals a likely widespread mechanism of energy conservation.

The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H/e ratio of 1.

Membrane-spanning electron transfer proteins from electrogenic bacteria: Production and investigation.

Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation.

Direct Prediction of EPR Spectra from Lipid Bilayers: Understanding Structure and Dynamics in Biological Membranes.

Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer.

Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.

Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin-cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors.

A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics.

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions.

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1.

Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.

Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals.

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated.

Development of a proteoliposome model to probe transmembrane electron-transfer reactions.

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decahaem cytochromes brought together inside a transmembrane porin to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system has been developed that contains Methyl Viologen as an internalized electron carrier and valinomycin as a membrane-associated cation exchanger. These proteoliposomes can be used as a model system to investigate MtrCAB function.

The 'porin-cytochrome' model for microbe-to-mineral electron transfer.

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved.

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration.

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources.

Structure of a bacterial cell surface decaheme electron conduit.

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages.

Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization.

Unlabelled: The iron responsive regulator Irr is found in a wide range of α-proteobacteria, where it regulates many genes in response to the essential but toxic metal iron. Unlike Fur, the transcriptional regulator that is used for iron homeostasis by almost all other bacterial lineages, Irr does not sense Fe(2+) directly, but, rather, interacts with a physiologically important form of iron, namely heme. Recent studies of Irr from the N(2)-fixing symbiont Rhizobium leguminosarum (Irr(Rl)) showed that it binds heme with submicromolar affinity at a His-Xxx-His (HxH) motif.

Prediction of nitroxide spin label EPR spectra from MD trajectories: application to myoglobin.

We report the prediction of motional EPR spectra of the metalloprotein sperm whale myoglobin spin labelled with nitroxide directly from Molecular Dynamics (MD) simulations at the atomistic scale. We show that an accurate simulation of EPR spectra can be achieved from a single MD trajectory which is in excellent agreement with experiment. Simulations have been carried out using a general method reported previously by us for the simulation of EPR spectra form single dynamical trajectories.

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme-copper site in cytochrome bo3, E. coli.

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo(3), a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T(1), of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2.

Heme-responsive DNA binding by the global iron regulator Irr from Rhizobium leguminosarum.

Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein.

Subunit organization in the TatA complex of the twin arginine protein translocase: a site-directed EPR spin labeling study.

The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy.

Electrochemical and EPR studies of two substituted bis-cadmium tris-phthalocyanine complexes: elucidation of unexpectedly different free-radical character.

The electronic and electron transfer behaviour of two examples of a recently discovered class of triple-decker sandwich complex based on three phthalocyanine ligands linked by two chelated cadmium ions has been investigated by EPR spectroscopy and cyclic voltammetry, square wave voltammetry and linear sweep voltammetry experiments. The two compounds, and , differ in the location of the eight alkyl groups attached to each of the phthalocyanine rings; at the non-peripheral sites in and the peripheral sites in . Quantitative comparison of the free radical character of and in solutions was undertaken by EPR spectroscopy and revealed that exists as a mixture of s = 0 and s = (1/2) species, whereas compound exists essentially as a spin (1/2) species alone.

The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion.

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster.

Signal perception by FNR: the role of the iron-sulfur cluster.

The metabolic flexibility of bacteria is key to their ability to survive and thrive in a wide range of environments. Optimal switching from one metabolic pathway to another is a key requirement for this flexibility. Respiration is a good example: many bacteria utilize O(2) as the terminal electron acceptor, but can switch to a range of other acceptors, such as nitrate, when O(2) becomes limiting.