Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

Magnetic circular dichroism (MCD) spectra, at ultraviolet–visible or near-infrared wavelengths (185–2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid

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

Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH–-ligated. The ferrous state is predominantly high-spin and, most likely, His/–. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form

African exceptions: democratic development in small island states

Institutions, Decisions and Collective Behaviou

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzyme

Thiosulfate dehydrogenases (TsdA) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic circular dichroism (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a micro-aerobe human pathogen, and from the purple sulfur bacterium Allochromatium vinosum. In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase respectively. The active site Heme 1 in both enzymes has His/Cys− ligation in the ferric and ferrous states and the midpoint potentials (Em) of the corresponding redox transformations are similar, −185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em, −129 mV) to His/Met (Em,+266 mV) but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s)

Denitrification bioreactor trial in the Russell River catchment of the Wet Tropics: final report

Dissolved inorganic nitrogen (DIN) in runoff from agricultural land is considered to have a significant detrimental impact on the Great Barrier Reef (GBR). Losses of DIN to runoff can be reduced by good agricultural practices, but they cannot be eliminated entirely in the Wet Tropics due to the need for adequate nitrogen supply to crops, the high solubility of DIN, particularly nitrate, and high rainfall. Thus, it is inevitable that DIN concentrations are higher in runoff from agricultural land than from forested areas. Some of this DIN is removed from the water as it moves through aquifers, creeks, rivers, and wetlands on its way to the sea, through the process of microbial denitrification. Denitrification involves the conversion of nitrate and nitrite (NOx-N) to dinitrogen (N2) gas, which is lost to the atmosphere. Denitrification requires NOx-N, organic matter, and low oxygen concentration. Wetlands provide these conditions, so DIN concentrations decline in water moving through them. Similarly, denitrifying bioreactors are designed to treat water by passing it through a porous organic material, typically woodchips. The woodchips provide organic matter for the microorganisms, which in turn lower the oxygen concentration, providing ideal conditions for denitrification. Denitrifying bioreactors are now widely used to remove the NOx-N component of DIN from agricultural runoff water elsewhere, but they have not yet been evaluated in the Wet Tropics. The Wet Tropics pose a challenge for efficacy due to the large volumes of water moving through the landscape. The objective of this project was “to establish the effectiveness of denitrifying bioreactors as a remediation technology for excess DIN in agricultural runoff within the Babinda Swamp Drainage Area (BSDA) of the Russell catchment”. The Russell River exports a disproportionate amount of DIN to the GBR lagoon because of the high rainfall and high proportion of agriculture, mostly sugarcane, in its catchment

DT‐PACE/ESHAP chemotherapy regimens as salvage therapy for multiple myeloma prior to autologous stem cell transplantation

Routine use of novel agents to treat newly diagnosed and relapsed multiple myeloma (MM) produces high response rates and improved survival. However, 15–20% of patients have suboptimal responses and their management remains challenging.1 Traditional regimens, such as DT‐PACE (dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, etoposide) and ESHAP (etoposide, methylprednisolone, cytarabine, cisplatin) are employed in patients with relapsed/refractory (RR) disease, and may bridge patients to autologous stem cell transplantation (ASCT).2-4 Originally developed to improve responses to traditional chemotherapy regimens, and enable stem cell mobilization,5-7 the role of infusional regimens in the context of novel agents is unclear, especially as recently reported series indicate relatively poor outcomes.8, 9 These regimens can be associated with significant toxicity,2 placing a burden on healthcare resources.10 We undertook a single‐centre retrospective analysis to assess the role of infusional regimens in RR MM patients to explore and identify features associated with clinical benefit. Relevant clinical information was obtained from electronic records. Overall response rate (ORR) and cytogenetic risk were assessed as per International Myeloma Working Group (IMWG) criteria (Table I).11 [Progression‐free (PFS) and overall survival (OS) were estimated using Kaplan–Meier and Cox regression methods (time‐dependent where appropriate)]