Duncan G. G. McMillan

Menaquinone-7 Is Specific Cofactor in Tetraheme Quinol Dehydrogenase CymA

Background: CymA is the central menaquinol-7 dehydrogenase in anaerobic respiration of Shewanella sp. Results: CymA uses menaquinone-7 as a cofactor. Conclusion: CymA has one cofactor site that is specific for menaquinone-7 and one low affinity Q/QH2 site that is in equilibrium with the quinone pool. Significance: The function of quinones needs to be reevaluated and crystallographically determined quinone binding pockets might not be the site of quinone conversion. Little is known about enzymatic quinone-quinol interconversions in the lipid membrane when compared with our knowledge of substrate transformations by globular enzymes. Here, the smallest example of a quinol dehydrogenase in nature, CymA, has been studied. CymA is a monotopic membrane tetraheme c-type cytochrome belonging to the NapC/NirT family and central to anaerobic respiration in Shewanella sp. Using protein-film electrochemistry, it is shown that vesicle-bound menaquinone-7 is not only a substrate for this enzyme but is also required as a cofactor when converting other quinones. Here, we propose that the high concentration of quinones in the membrane negates the evolutionary pressure to create a high affinity active site. However, the instability and reactivity of reaction intermediate, semiquinone, might require a cofactor that functions to minimize damaging side reactions.

Concentrating membrane proteins using asymmetric traps and AC electric fields.

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a nested trap and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins.

A Specific Adaptation in the a Subunit of Thermoalkaliphilic F1FO-ATP Synthase Enables ATP Synthesis at High pH but Not at Neutral pH Values

Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. TA2.A1 numbering) within the a subunit of these F1Fo-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F1Fo) was developed in Escherichia coli DK8 (Δatp). Amino acid substitutions were made in the a subunit of TA2F1Fo at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus Pi-loaded right-side-out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F1Fo and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0–9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH values 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue Lys180 is a specific adaptation within the a subunit of TA2F1Fo to facilitate proton capture at high pH. At pH values near the pKa of Lys180, the trapped protons readily dissociate to reach the subunit c binding sites, but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H+ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited.

Protein–Protein Interaction Regulates the Direction of Catalysis and Electron Transfer in a Redox Enzyme Complex

Protein–protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein–protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis. We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein–protein interactions.

Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase

Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly other HCOs, to maintain a suitable ΔpH under extreme redox conditions.

Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation.

F1F0 ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acidin/alkalineout). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity, which can be relieved in the presence of lauryldimethylamine oxide. Here, we characterized the rotary dynamics of the Caldalkalibacillus thermarum TA2.A1 F1 ATPase (TA2F1) with two forms of single molecule analysis, a magnetic bead duplex and a gold nanoparticle. TA2F1 rotated in a counterclockwise direction in both systems, adhering to Michaelis-Menten kinetics with a maximum rotation rate (Vmax) of 112.4 revolutions/s. TA2F1 displayed 120° unitary steps coupled with ATP hydrolysis. Torque measurements revealed the highest torque (52.4 piconewtons) derived from an F1 molecule using fluctuation theorem. The implications of high torque in terms of extreme environment adaptation are discussed.

Population Changes in a Community of Alkaliphilic Iron-Reducing Bacteria Due to Changes in the Electron Acceptor: Implications for Bioremediation at Alkaline Cr(VI)-Contaminated Sites

A serial enrichment culture has been grown in an alkaline Fe(III)-citrate-containing medium from an initial inoculum from a soil layer beneath a chromium ore processing residue (COPR) disposal site where Cr(III) is accumulating from Cr(VI) containing leachate. This culture is dominated by two bacterial genera in the order Clostridiales, Tissierella, and an unnamed Clostridium XI subgroup. This paper investigates the growth characteristics of the culture when Cr(VI) is added to the growth medium and when aquifer sand is substituted for Fe(III)-citrate. The aim is to determine how the availability and chemical form of Fe(III) affects the growth of the bacterial consortium, to determine the impact of Cr(VI) on growth, and thus attempt to understand the factors that are controlling Cr(III) accumulation beneath the COPR site. The culture can grow fermentatively at pH 9.2, but growth is stronger when it is associated with Fe(III) reduction. It can withstand Cr(VI) in the medium, but growth only occurs once Cr(VI) is removed from solution. Cr(VI) reduced the abundance of Tissierella sp. in the culture, whereas the Clostridium XI sp. was Cr(VI) tolerant. In contrast, growth with solid phase Fe(III)-oxyhydroxides (present as coatings on aquifer sand) favoured the Tissierella C sp., possibly because it produces riboflavin as an extracellular electron shuttling compound allowing more efficient electron transfer to solid Fe(III) phases. Thus, it is suggested that bacterially mediated Cr(III) reduction in the soil beneath the COPR site is dependent on Fe(III) reduction to sustain the bacterial community.

Catalytic robustness and torque generation of the F1-ATPase

The F1-ATPase is the catalytic portion of the FoF1 ATP synthase and acts as a rotary molecular motor when it hydrolyzes ATP. Two decades have passed since the single-molecule rotation assay of F1-ATPase was established. Although several fundamental issues remain elusive, basic properties of F-type ATPases as motor proteins have been well characterized, and a large part of the reaction scheme has been revealed by the combination of extensive structural, biochemical, biophysical, and theoretical studies. This review is intended to provide a concise summary of the fundamental features of F1-ATPases, by use of the well-described model F1 from the thermophilic Bacillus PS3 (TF1). In the last part of this review, we focus on the robustness of the rotary catalysis of F1-ATPase to provide a perspective on the re-designing of novel molecular machines.

Electrochemical stability of the polymer-derived nitrogen-doped carbon: an elusive goal?

Nitrogen-doped carbon is a promising metal-free catalyst for oxygen reduction reaction in fuel cells and metal-air batteries. However, its practical application necessitates a significant cost reduction, which can be achieved in part by using new synthetic methods and improvement of catalytic activity by increasing the density of redox active centers. This can be modulated by using polymer as the carbon and nitrogen sources. Although, superior catalytic activity of such N-doped C has been investigated in details, the electrochemical long-term stability of polymer-derived doped-carbon is still unclear. Herein, in this study we generated N-doped carbon from the most recommended polymer that is comparable to the state-of-the-art materials with porosity as high as 2,086xa0m2xa0g−1 and a nitrogen doping level of 3–4xa0at.%, of which 56xa0% is pyrrolic N, 36.1xa0% pyridinic and ~8xa0% graphitic. The electrochemical characterization shows that N-doped carbon is catalytic toward oxygen reduction in an alkaline electrolyte via a favorable four-electron process, however, not stable under long-term potential scanning. The irreversible electrochemical oxidation of this material is associated with the presence of a significant content or pyrrolic and pyridinic N close to the edge of the carbon network originating from the polypyrrole precursor. These structures are less stable under operating electrochemical potential. The role of polypyrrole as the precursor of N-doped carbons has to be carefully revised since it supplies sufficient number of catalytic sites, but also generates unstable functionalities on the carbon surface.