Opportunities for mesoporous nanocrystalline SnO2 electrodes in kinetic and catalytic analyses of redox proteins

PFV (protein film voltammetry) allows kinetic analysis of redox and coupled-chemical events. However, the voltammograms report on the electron transfer through a flow of electrical current such that simultaneous spectroscopy is required for chemical insights into the species involved. Mesoporous nanocrystalline SnO2 electrodes provide opportunities for such ‘spectroelectrochemical’ analyses through their high surface area and optical transparency at visible wavelengths. Here, we illustrate kinetic and mechanistic insights that may be afforded by working with such electrodes through studies of Escherichia coli NrfA, a pentahaem cytochrome with nitrite and nitric oxide reductase activities. In addition, we demonstrate that the ability to characterize electrocatalytically active protein films by MCD (magnetic circular dichroism) spectroscopy is an advance that should ultimately assist our efforts to resolve catalytic intermediates in many redox enzymes

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H2O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately −240 mV) and low-spin (approximately −110, −190 and −265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em=−80 mV) in the presence of NADH (Em=−320 mV) and an NADH–menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.</jats:p

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 Discovery2006, 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. Nature1977, 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

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity–potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity–potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity–potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies

Electrode assemblies composed of redox cascades from microbial respiratory electron transfer chains

Respiratory and photosynthetic electron transfer chains are dependent on vectorial electron transfer through a series of redox proteins. Examples include electron transfer from NapC to NapAB nitrate reductase in Paracoccus denitrificans and from CymA to Fcc3 (flavocytochrome c3) fumarate reductase in Shewanella oneidensis MR-1. In the present article, we demonstrate that graphite electrodes can serve as surfaces for the stepwise adsorption of NapC and NapAB, and the stepwise adsorption of CymA and Fcc3. Aspects of the catalytic properties of these assemblies are different from those of NapAB and Fcc3 adsorbed in isolation. We propose that this is due to the formation of NapC-NapAB and of CymA-Fcc3 complexes that are capable of supporting vectorial electron transfer

Protein voltammetry and spectroscopy: integrating approaches

Cyclic voltammetry readily visualizes the redox properties of many proteins. Net electron exchange between the protein and an electrode produces an electrical current that simultaneously quantitates and characterizes the underlying redox event(s). However, no direct information regarding the molecular origin, or consequences, of electron transfer is available. Integrating voltammetric and spectroscopic methods is one route to a more ‘holistic’ description of protein electron transfer. Here, we illustrate this approach with spectroelectrochemical studies of Rhodovulum sulfidophilum cytochrome c 2 and Escherichia coli cytochrome bd that employ electronic absorbance, infra-red and magnetic circular dichroism spectroscopies

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme–copper site in cytochrome bo 3, 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. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T 2, of the dinuclear coupled pair Fe(III)–CuB(II) in the oxidized form of QO that is too short to measure directly. In the case of the F' state, the relaxation properties of the heme–copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and CuB(II) coupled by approximately 1.5–3 cm-1 to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine–histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements

Spectroelectrochemical Characterization of a Pentaheme Cytochrome in Solution and as Electrocatalytically Active Films on Nanocrystalline Metal-Oxide Electrodes

The pentaheme containing cytochrome, NrfA, from Escherichia coli catalyzes the six-electron reduction of nitrite and the five-electron reduction of nitric oxide. Crystallographic and spectroscopic studies have provided a structural framework for these mechanisms. The active site includes a high-spin heme, and four low-spin, bis-his coordinated hemes are positioned to facilitate intra- and intermolecular electron exchange. However, despite the use of protein film voltammetry to provide kinetic descriptions of NrfA catalysis at graphite and gold electrodes, the thermodynamic descriptions of heme redox activity remain incomplete. Here we rectify this situation with the observation of nonturnover signals from NrfA adsorbed on mesoporous SnO2 electrodes. Simultaneous cyclic voltammetry and electronic absorption spectroscopy define reduction potentials for the high- and low-spin hemes. These reduction potentials are shown to be similar to those exhibited by the enzyme in solution and defined by electrodic reduction monitored by magnetic circular dichroism. Thus, NrfA is shown to undergo minimal perturbation of its electronic and thermodynamic properties on adsorption giving confidence to correlations of properties deduced from various methods and in approaches that may well facilitate studies of other oxidoreductases where catalytic protein film voltammetry is well-defined but nonturnover signals elusive