Anaerobic thin-layer electrochemical cell for planar optically transparent electrodes

Bowden, Edmond F.; Cohen, David J.; Hawkridge, Fred M.

doi:10.1021/ac00243a040

Cited by 17 publications

(12 citation statements)

References 22 publications

(28 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This cell was similar to a previous design (41). A nominal optical pathlength of 0.1 mm was realized using 0.1 mm thick Teflon coated fiberglass tape (Small Parts, Inc.).…”

Section: Methodsmentioning

confidence: 69%

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

et al. 2011

Self Cite

View full text Add to dashboard Cite

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine-86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D (PDB 3MYN) and M86E (PDB 3MYM) mutants reveals the structural origins of a stable catalytic triad in DHP A. A decrease in rate of H2O2 activation, as well as a lowered reduction potential than the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine to bind to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is 5-coordinate and this resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a 6-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a 6-coordinate low-spin adduct, which inhibits the ligation and activation of H2O2 as required for peroxidase activity. The results suggest that the role of the proximal charge relay in peroxidases regulates the redox potential of the heme Fe, but that the trans effect is a carefully balanced property that can both activate H2O2 and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, three mutants in the M86 position, M86A, M86D, and M86E were studied by spectroelectrochemistry and NMR spectroscopy of 13C15N -labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the Nδ hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.

show abstract

“…This cell was similar to a previous design (41). A nominal optical pathlength of 0.1 mm was realized using 0.1 mm thick Teflon coated fiberglass tape (Small Parts, Inc.).…”

Section: Methodsmentioning

confidence: 69%

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…SEC was carried out in a previously designed anaerobic UV-visible spectroelectrochemical cell made of cast acrylic (Small Parts, Inc.) that utilized an optically-transparent thin-layer electrode (OTTLE) of antimony-tin oxide (ATO, Delta Technologies, Limited) (44). The pathlength for the cell was maintained at 0.1 mm by use of Teflon coated fiberglass tape (Small Parts, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata

et al. 2010

Self Cite

View full text Add to dashboard Cite

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UVvisible, resonance Raman and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system. Figure SD1); UVvisible spectra of the (tri)halophenol complexes of DHP B ( Figure SD2); UV-visible spectroscopic monitoring of the oxidative dehalogenation of trihalophenols as catalyzed by DHP B in the presence of hydrogen peroxide ( Figure SD3); dependence of k obs for the reaction between ferric DHP B with hydrogen peroxide (2.5 -25 equivalents) at pH 7 yielding Compound ES ( Figure SD4); stopped-flow UV-visible spectroscopic monitoring of ( Figure SD5), and DCQ product formation and TCP co-substrate loss for ( Figure SD6), the double-mixing reaction between preformed DHP B Compound ES and TCP at pH 7; stopped-flow UV-visible spectroscopic monitoring of ( Figure SD7), and DCQ product formation and TCP co-substrate loss for ( Figure SD8), the doublemixing reaction between ferric DHP B pre-incubated with TCP for 500 ms prior to its reaction with a 10-fold excess of H 2 O 2 (in situ generated Compound ES) at pH 7; stopped-flow UV-visible spectroscopic monitoring of the double-mixing reaction between ferric DHP B pre-incubated with a 7-fold molar excess of DCQ for 500 ms prior to its reaction with a 2.5-fold excess of H 2 O 2 (in situ generated Compound ES) ( Figure SD9); stopped-flow UV-visible spectroscopic monitoring of the reaction between ferric DHP B and a 7-fold excess of DCQ at pH 7 ( Figure SD10); reduction of Compound RH yielding Compound P 426 ( Figure SD11), reduction of Compound RH yielding oxyferrous DHP B ( Figure SD12). This m...

show abstract

“…Spectroelectrochemical techniques have been applied mostly in connection with optical or EPR studies. For IR spectroscopy, several cell designs have been reported either for transmission (Heineman et al, 1968) or reflection techniques (Hansen et al, 1966), or those intended for the optical region, but which can be adapted for the IR by the use of appropriate windows (Anderson et al, 1979;Murray et al, 1967;Bowden et al, 1982). Dispersive IR spectroscopy was used in earlier reports to monitor stable electrolysis products.…”

Section: Introductionmentioning

confidence: 99%

FOURIER TRANSFORM INFRARED SPECTROELECTROCHEMISTRY OF THE BACTERIOCHLOROPHYLL a ANION RADICAL

Mäntele

Wollenweber

Rashwan

et al. 1988

Photochem & Photobiology

View full text Add to dashboard Cite

Abstract— Fourier transform infrared (FTIR) difference spectroscopy of the electrochemically generated anion radical of bacteriochlorophyll a was used to follow the molecular changes upon one‐electron reduction. An IR spectroelectrochemical cell was constructed, allowing in situ electrolysis in connection with spectroscopic investigations from 200 to 10 000 nm. FTIR difference spectra of the BChl a anion formation in THF d8 at U=+0.9 V (as determined by ferrocene calibration) were obtained. After complete formation of the radical, the reverse process was followed. Comparison of visible and IR spectra of the reduction and re‐oxidation processes indicates that more than 90% of the BChl a anion could be formed with 90% of it being reversible. The main IR absorbance changes are observed for the conjugated and even for the non‐conjugated C=O groups of BChl a. These results demonstrate the use of the combination of FTIR spectroscopy and electrochemistry for the characterization of radicals of the isolated pigments and of their in vivo bonding to the protein environment.

show abstract

Anaerobic thin-layer electrochemical cell for planar optically transparent electrodes

Cited by 17 publications

References 22 publications

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata

FOURIER TRANSFORM INFRARED SPECTROELECTROCHEMISTRY OF THE BACTERIOCHLOROPHYLL a ANION RADICAL

Contact Info

Product

Resources

About