Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs.

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK a ≈ 7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V max and the nitrite dependence of the catalytic current-potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.

Integrated systems for rapid point of care (PoC) blood cell analysis.

Counting the different subpopulations of cells in a fingerprick of human blood is important for a number of clinical point-of-care (PoC) applications. It is a challenge to demonstrate the integration of sample preparation and detection techniques in a single platform. In this paper we demonstrate a generic microfluidic platform that combines sample processing and characterisation and enumeration in a single, integrated system. Results of microfluidic 3-part differential leukocyte (granulocyte, lymphocyte and monocyte) counts, together with erythrocyte and thrombocyte (platelet) counts, in human blood are shown and corroborated with results from hospital clinical laboratory analysis.

Effect of the structure of cholesterol-based tethered bilayer lipid membranes on ionophore activity.

Tethered bilayer lipid membranes (tBLM) are formed on 1) pure tether lipid triethyleneoxythiol cholesterol (EO(3)C) or on 2) mixed self-assembled monolayers (SAMs) of EO(3)C and 6-mercaptohexanol (6MH). While EO(3)C is required to form a tBLM with high resistivity, 6MH dilutes the cholesterol content in the lower leaflet of the bilayer forming ionic reservoirs required for submembrane hydration. Here we show that these ionic reservoirs are required for ion transport through gramicidin or valinomycin, most likely due to the thermodynamic requirements of ions to be solvated once transported through the membrane. Unexpectedly, electrochemical impedance spectroscopy (EIS) shows an increase of capacitance upon addition of gramicidin, while addition of valinomycin decreases the membrane resistance in the presence of K(+) ions. We hypothesise that this is due to previously reported phase separation of EO(3)C and 6MH on the surface. This results in ionic reservoirs on the nanometre scale, which are not fully accounted for by the equivalent circuits used to describe the system.

Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry.

Miniature high speed label-free cell analysis systems have yet to be developed, but have the potential to deliver fast, inexpensive and simple full blood cell analysis systems that could be used routinely in clinical practice. We demonstrate a microfluidic single cell impedance cytometer that performs a white blood cell differential count. The device consists of a microfluidic chip with micro-electrodes that measure the impedance of single cells at two frequencies. Human blood, treated with saponin/formic acid to lyse erythrocytes, flows through the device and a complete blood count is performed in a few minutes. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from CD antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Tests with patient samples showed 95% correlation against commercial (optical/Coulter) blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.

Voltammetry and in situ scanning tunneling microscopy of cytochrome C nitrite reductase on Au(111) electrodes.

Escherichia coli cytochrome c nitrite reductase (NrfA) catalyzes the six-electron reduction of nitrite to perform an important role in the biogeochemical cycling of nitrogen. Here we describe NrfA adsorption on single-crystal Au(111) electrodes as an electrocatalytically active film in which the enzyme undergoes direct electron exchange with the electrode. The adsorbed NrfA has been imaged to molecular resolution by in situ scanning tunneling microscopy (in situ STM) under full electrochemical potential control and under conditions where the enzyme is electrocatalytically active. Details of the density and orientational distribution of NrfA molecules are disclosed. The submonolayer coverage resolved by in situ STM is readily reconciled with the failure to detect nonturnover signals in cyclic voltammetry of the NrfA films. The molecular structures show a range of lateral dimensions. These are suggestive of a distribution of orientations that could account for the otherwise anomalously low turnover number calculated for the total population of adsorbed NrfA molecules when compared with that determined for solutions of NrfA. Thus, comparison of the voltammetric signals and in situ STM images offers a direct approach to correlate electrocatalytic and molecular properties of the protein layer, a long-standing issue in protein film voltammetry.

Diode or tunnel-diode characteristics? Resolving the catalytic consequences of proton coupled electron transfer in a multi-centered oxidoreductase.

Protein film voltammetry has been employed to define multiple catalytic consequences of proton coupled electron transfer (PCET) in a cytochrome c nitrite reductase. Current-potential profiles reflecting the steady-state rate of nitrite-limited reduction have been defined from pH 4 to 8. Lowering the electrode potential at pH 8 causes the catalytic current to increase and then decrease before it takes a value independent of any further lowering of electrode potential. By comparison, at pH 4, catalysis is initiated at more positive electrode potentials in an approximately sigmoidal fashion with no attenuation of the catalytic rate evident at more negative electrode potentials. The results show that activity is turned on by the coupled transfer of two electrons and one proton to the enzyme. The decreased rate of catalysis at lower electrode potentials under more alkaline conditions shows that this rate attenuation occurs only when reduction is not coupled to compensating protonation(s) of the enzyme. Sites within the enzyme whose reduction and/or protonation may contribute to the definition of these activities are discussed.

Resolving complexity in the interactions of redox enzymes and their inhibitors: contrasting mechanisms for the inhibition of a cytochrome c nitrite reductase revealed by protein film voltammetry.

Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium. The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV). Azide inhibition is rapid and reversible. Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (approximately 40 mM) with a lower affinity than to the enzyme alone (approximately 15 mM) and leads to complete inhibition of enzyme activity. The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (approximately 39 microM) than when it is reduced. By contrast, binding and release of cyanide are sluggish. The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme. Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme. The biscyano complex is inactive. These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme.

Redox-triggered events in cytochrome c nitrite reductase.

Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.