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Lovett B.W.,Heriot - Watt University | Harmer J.,Center for Advanced Electronic Spin Resonance
Journal of Magnetic Resonance | Year: 2012

Over approximately the last 15 years the electron paramagnetic resonance (EPR) technique of double electron electron resonance (DEER) has attracted considerable attention since it allows for the precise measurement of the dipole-dipole coupling between radicals and thus can lead to distance information between pairs of radicals separated by up to ca. 8 nm. The "deadtime free" 4-pulse DEER sequence is widely used but can suffer from poor sensitivity if the electron spin-echo decays too quickly to allow collection of a sufficiently long time trace. In this paper we present a method which takes advantage of the much greater sensitivity that the 3-pulse sequence offers over the 4-pulse sequence since the measured electron spin-echo intensity (for equal sequence lengths) is greater. By combining 3- and 4-pulse DEER time traces using a method coined DEER-Stitch (DEERS) accurate dipole-dipole coupling measurements can be made which combine the sensitivity of the 3-pulse DEER sequence with the deadtime free advantage of the 4-pulse DEER sequence. To develop the DEER-Stitch method three systems were measured: a semi-rigid bis-nitroxide labeled nanowire, the bis-nitroxide labeled protein CD55 with a distance between labels of almost 8 nm and a dimeric copper amine oxidase from Arthrobacter globiformis (AGAO). © 2012 Elsevier Inc. All rights reserved.

Neil S.R.T.,University of Oxford | Maeda K.,University of Oxford | Maeda K.,Center for Advanced Electronic Spin Resonance | Henbest K.B.,University of Oxford | And 6 more authors.
Molecular Physics | Year: 2010

Cavity enhanced absorption spectroscopy (CEAS) combined with phase-sensitive detection is employed to study the effects of static magnetic fields on radical recombination reactions. The chemical system comprises the photochemically generated thionine semiquinone radical and a 1,4-diazabicyclo[2.2.2]octane (DABCO) cationic radical in a micellar solution of sodium dodecyl sulphate. Data obtained using the modulated CEAS technique, describing the magnetic field effect (MFE) on reaction yields, are shown to be superior to those obtained using conventional transient absorption (TA) flash photolysis methods typically employed for these measurements. The high sensitivity afforded by modulated CEAS detection is discussed in terms of the new possibilities it offers such as the measurement of magnetic field effects in real biological systems which have hitherto been largely beyond the detection capabilities of existing techniques. © 2010 Taylor & Francis.

Evans R.M.,South Parks Road | Parkin A.,South Parks Road | Roessler M.M.,South Parks Road | Roessler M.M.,Center for Advanced Electronic Spin Resonance | And 8 more authors.
Journal of the American Chemical Society | Year: 2013

"Hyd-1", produced by Escherichia coli, exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H2 oxidation activity in the presence of O2 -an intruder that normally incapacitates the sulfur-and electron-rich active site. The mechanism of "O2 tolerance" involves a critical role for the Fe-S clusters of the electron relay, which is to ensure the availability -for immediate transfer back to the active site -of all of the electrons required to reduce an attacking O2 molecule completely to harmless H 2O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 Å from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H2 oxidation activity in 1% O 2. The [3Fe-4S] cluster is essential only for the first stage of complete O2 reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state "Ni-B" or "Ready" (Ni(III)-OH). Potentiometric titrations show that Ni-B is easily reduced (Em ≈ +0.1 V at pH 6.0); this final stage of the O 2-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme's response to O2, viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H2 oxidation activity; thus, O 2 behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe-S clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase. © 2013 American Chemical Society.

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