Translocating kilobase RNA through the Staphylococcal α-hemolysin nanopore.

The electrophoretic translocation of polynucleotides through nanopores may permit direct single-molecule nucleic acid sequencing. Here we describe the translocation of ssRNA heteropolymers (91-6083 bases) through the α-hemolysin nanopore. Translocation of these long ssRNAs is characterized by surprisingly long, almost complete ionic current blockades with durations averaging milliseconds per base (at +180 mV). The event durations decrease exponentially with increased transmembrane potential but are largely unaffected by the presence of urea. When the ssRNA is coupled at the 3' end to streptavidin, which cannot translocate through the pore, permanent blockades are observed, supporting our conclusion that the transient blockades arise from ssRNA translocation.

Bilirubin oxidase from Myrothecium verrucaria: X-ray determination of the complete crystal structure and a rational surface modification for enhanced electrocatalytic O2 reduction.

The blue multi-copper oxidase bilirubin oxidase (BOx) from the ascomycete plant pathogen Myrothecium verrucaria (Mv) efficiently catalyses the oxidation of bilirubin to biliverdin, with the concomitant reduction of O(2) to water, a reaction of considerable interest for low-temperature bio-fuel cell applications. We have solved the complete X-ray determined structure of Mv BOx at 2.4 Å resolution, using molecular replacement with the Spore Coat Protein A (CotA) enzyme from Bacillus subtilis (PDB code 1GSK) as a template. The structure reveals an unusual environment around the blue type 1 copper (T1 Cu) that includes two non-coordinating hydrophilic amino acids, asparagine and threonine. The presence of a long, narrow and hydrophilic pocket near the T1 Cu suggests that structure of the substrate-binding site is dynamically determined in vivo. We show that the interaction between the binding pocket of Mv BOx and its highly conjugated natural organic substrate, bilirubin, can be used to stabilise the enzyme on a pyrolytic graphite electrode, more than doubling its electrocatalytic activity relative to the current obtained by simple adsorption of the protein to the carbon surface.

Gas pressure effects on the rates of catalytic H(2) oxidation by hydrogenases.

During catalysis by hydrogenases, entities no larger than H(2) or H(+) reach and leave a deeply buried active site, by as yet unidentified pathways. Novel experiments, conducted mainly with the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha, explore why small excess gas pressures (H(2) or He) attenuate the rate of H(2) oxidation.

A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases.

In biology, rapid oxidation and evolution of H(2) is catalyzed by metalloenzymes known as hydrogenases. These enzymes have unusual active sites, consisting of iron complexed by carbonyl, cyanide, and thiolate ligands, often together with nickel, and are typically inhibited or irreversibly damaged by O(2). The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H(2) as an energy source with O(2) as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an "O(2)-tolerant" hydrogenase. The mechanism of O(2) tolerance of Re MBH has been probed by measuring H(2) oxidation activity in the presence of O(2) over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O(2) and subsequent reactivation of the active site. Most significantly, O(2) tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H(2) affinity or the kinetics of O(2) attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O(2) tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases.

Oxygen-tolerant H2 oxidation by membrane-bound [NiFe] hydrogenases of ralstonia species. Coping with low level H2 in air.

Knallgas bacteria such as certain Ralstonia spp. are able to obtain metabolic energy by oxidizing trace levels of H2 using O2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H2 in the presence of O2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O2 tolerance, we conducted a study on the membrane-bound hydrogenase from Ralstonia eutropha H16 and that of the closely related organism Ralstonia metallidurans CH34, which was purified using a new heterologous overproduction system. Direct electrochemical methods were used to determine apparent inhibition constants for O2 inhibition of H2 oxidation (K I(app)O2) for each enzyme. These values were at least 2 orders of magnitude higher than those of "standard" [NiFe] hydrogenases. Amino acids close to the active site were exchanged in the membrane-bound hydrogenase of R. eutropha H16 for those from standard hydrogenases to probe the role of individual residues in conferring O2 sensitivity. Michaelis constants for H2 (K M H2) were determined, and for some mutants these were increased more than 20-fold relative to the wild type. Mutations resulting in membrane-bound hydrogenase enzymes with increased K M H2 or decreased K I(app)O2 values were associated with impaired lithoautotrophic growth in the presence of high O2 concentrations.

Dynamic electrochemical investigations of hydrogen oxidation and production by enzymes and implications for future technology.

This tutorial review describes studies of hydrogen production and oxidation by biological catalysts--metalloenzymes known as hydrogenases--attached to electrodes. It explains how the electrocatalytic properties of hydrogenases are studied using specialised electrochemical techniques and how the data are interpreted to allow assessments of catalytic rates and performance under different conditions, including the presence of O2, CO and H2S. It concludes by drawing some comparisons between the enzyme active sites and platinum catalysts and describing some novel proof-of-concept applications that demonstrate the high activities and selectivities of these 'alternative' catalysts for promoting H2 as a fuel.

Hydrogen production under aerobic conditions by membrane-bound hydrogenases from Ralstonia species.

Studies have been carried out to establish the ability of O2-tolerant membrane-bound [NiFe] hydrogenases (MBH) from Ralstonia sp. to catalyze H2 production in addition to H2 oxidation. These hydrogenases are not noted for H2-evolution activity, and this is partly due to strong product inhibition. However, when adsorbed on a rotating disk graphite electrode the enzymes produce H2 efficiently, provided the H2 product is continuously removed by rapidly rotating the electrode and flowing N2 through the gastight electrochemical cell. Electrocatalytic H2 production proceeds with minimal overpotentiala significant observation because lowering the overpotential (the electrochemically responsive activation barrier) is seen as crucial in developing small-molecule catalysts for H2 production. A mutant having a high KM for H2 oxidation did not prove to be a better H2 producer relative to the wild type, thus suggesting that weak binding of H2 does not itself confer a tendency to be a H2 producer. Inhibition by H2 is much stronger than inhibition by CO and, most significantly, even O2. Consequently, H2 can be produced sustainably in the presence of O2 as long as the H2 is removed continuously, thereby proving the feasibility for biological H2 production in air.

Electricity from low-level H2 in still air--an ultimate test for an oxygen tolerant hydrogenase.

We demonstrate an extreme test of O(2) tolerance for a biological hydrogen-cycling catalyst: the generation of electricity from just 3% H(2) released into still, ambient air using an open fuel cell comprising an anode modified with the unusual hydrogenase from Ralstonia metallidurans CH34, that oxidizes trace H(2) in atmospheric O(2), connected via a film of electrolyte to a cathode modified with the fungal O(2) reductase, laccase.

Electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels.

Use of hydrogen in fuel cells requires catalysts that are tolerant to oxygen and are able to function in the presence of poisons such as carbon monoxide. Hydrogen-cycling catalysts are widespread in the bacterial world in the form of hydrogenases, enzymes with unusual active sites composed of iron, or nickel and iron, that are buried within the protein. We have established that the membrane-bound hydrogenase from the beta-proteobacterium Ralstonia eutropha H16, when adsorbed at a graphite electrode, exhibits rapid electrocatalytic oxidation of hydrogen that is completely unaffected by carbon monoxide [at 0.9 bar (1 bar = 100 kPa), a 9-fold excess] and is inhibited only partially by oxygen. The practical significance of this discovery is illustrated with a simple fuel cell device, thus demonstrating the feasibility of future hydrogen-cycle technologies based on biological or biologically inspired electrocatalysts having high selectivity for hydrogen.

Hydrogen cycling by enzymes: electrocatalysis and implications for future energy technology.

Hydrogenases provide an inspiration for future energy technologies. The active sites of these microbial enzymes contain Fe or Ni and Fe coordinated by CO and CN ligands: yet they have activities for hydrogen cycling that compare with Pt catalysts. Is there a future for enzymes in technological H2 cycling? There are obviously going to be disadvantages, perhaps overwhelming, as enzymes are notoriously fragile; yet what are the positive aspects and can we learn any chemistry that might be applied to produce the electrolytic and fuel cell catalysts of the future? We have developed a suite of novel electrochemical experiments to probe the chemistry of hydrogenases. The reactions are controlled and monitored at the surface of a small electrode, and characteristic catalytic properties are discernible from tiny amounts of sample material, so this approach can be used to search the microbial world for the best catalysts. Although electrochemistry does not provide structural information directly, it does give a "road map" by which to navigate the pathways and conditions that lead to particular states of the enzymes. This has prompted many interdisciplinary collaborations with other scientists who have provided microbiological, spectroscopic and structural contexts for this work. This article describes how these electrochemical experiments are set up, the data are analysed, and the results interpreted. We have determined mechanisms of catalysis, electron transfer, activation and inactivation, and defined important properties such as O2 tolerance and CO resistance in physical terms. Using an O2-tolerant hydrogenase, we have demonstrated a "proof of concept" miniature fuel cell that will run on a mixed H2/O2 feed in aqueous solution.