8 research outputs found
Proton transfer in the quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus during reduction of oxygen
Bacterial nitric oxide reductases (NOR) are integral membrane proteins that
catalyse the reduction of nitric oxide to nitrous oxide, often as a step in
the process of denitrification. Most functional data has been obtained with
NORs that receive their electrons from a soluble cytochrome c in the periplasm
and are hence termed cNOR. Very recently, the structure of a different type of
NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus
stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha,
A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat.
Struct. Mol. Biol. 19 (2012) 238–246]. In this study, we have investigated the
reaction between this qNOR and oxygen. Our results show that, like some cNORs,
the G. stearothermophilus qNOR is capable of O2 reduction with a turnover of ~
3 electrons s− 1 at 40 °C. Furthermore, using the so-called flow-flash
technique, we show that the fully reduced (with three available electrons)
qNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that
oxidises the low-spin heme b. This reaction is coupled to proton uptake from
solution and presumably forms a ferryl intermediate at the active site. The pH
dependence of the reaction is markedly different from a corresponding reaction
in cNOR from Paracoccus denitrificans, indicating that possibly the proton
uptake mechanism and/or pathway differs between qNOR and cNOR. This study
furthermore forms the basis for investigation of the proton transfer pathway
in qNOR using both variants with putative proton transfer elements modified
and measurements of the vectorial nature of the proton transfer. This article
is part of a Special Issue entitled: 17th European Bioenergetics Conference
(EBEC 2012)
Proton transfer in the quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus during reduction of oxygen
Bacterial nitric oxide reductases (NOR) are integral membrane proteins that
catalyse the reduction of nitric oxide to nitrous oxide, often as a step in
the process of denitrification. Most functional data has been obtained with
NORs that receive their electrons from a soluble cytochrome c in the periplasm
and are hence termed cNOR. Very recently, the structure of a different type of
NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus
stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha,
A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat.
Struct. Mol. Biol. 19 (2012) 238–246]. In this study, we have investigated the
reaction between this qNOR and oxygen. Our results show that, like some cNORs,
the G. stearothermophilus qNOR is capable of O2 reduction with a turnover of ~
3 electrons s− 1 at 40 °C. Furthermore, using the so-called flow-flash
technique, we show that the fully reduced (with three available electrons)
qNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that
oxidises the low-spin heme b. This reaction is coupled to proton uptake from
solution and presumably forms a ferryl intermediate at the active site. The pH
dependence of the reaction is markedly different from a corresponding reaction
in cNOR from Paracoccus denitrificans, indicating that possibly the proton
uptake mechanism and/or pathway differs between qNOR and cNOR. This study
furthermore forms the basis for investigation of the proton transfer pathway
in qNOR using both variants with putative proton transfer elements modified
and measurements of the vectorial nature of the proton transfer. This article
is part of a Special Issue entitled: 17th European Bioenergetics Conference
(EBEC 2012)
Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics
The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods