Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster’s Blue in methanol dehydrogenase assays

Abstract

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented.

Role of phenazine‐enzyme physiology for current generation in a bioelectrochemical system

“…An enzyme assay was performed with the purified proteins using PCA and PYO as prospective electron acceptors. The standard electron acceptors of an established dehydrogenase enzyme assay (Jahn et al, 2020), phenazine methanosulfate (PMS) and dichlorophenolindophenol (DCPIP), were used as control reaction to verify activity of the enzyme. When either PCA or PYO was used as electron acceptor in this reaction, the Gcd of P. aeruginosa PA14 was able to catalyse the transfer of electrons from glucose to these phenazines, which resulted in their measurable reduction (Fig.…”

Neodymium as Metal Cofactor for Biological Methanol Oxidation: Structure and Kinetics of an XoxF1-Type Methanol Dehydrogenase

“…The XoxF MDHs can be divided into those with a relatively high affinity (0.8 to 3.6 μM methanol) of types XoxF1 and XoxF2 (including the XoxF1 studied here) ( 28 , 33 , 46 ), and those with a relatively low affinity (29 to 55 μM methanol) of types XoxF4 and XoxF5 ( 68 – 70 ). Still, the conditions for the activity assays used for different XoxF-type MDHs vary greatly in addition to experimental difficulties, such as background reactions and instability of the artificial electron acceptors ( 71 ). Hence, values can often not be directly compared.…”

“…Methanol dehydrogenase activity tests were performed in 100 mM multicomponent buffer (25 mM citric acid, 25 mM bis-Tris, 25 mM Tris, and 25 mM N -cyclohexyl-2-aminoethanesulfonic acid [CHES]), 1 mM phenazine ethosulfate (PES), and 100 μM 2,6-dichlorophenolindophenol (DCPIP) at pH 7 and 45°C in an Epoch 2 plate reader and 96-well plates ( 71 ). Each well contained 200 μl total volume.…”

Harnessing methylotrophs as a bacterial platform to reduce adverse effects of the use of the heavy lanthanide gadolinium in magnetic resonance imaging

“…Ghosh and Quayle 1979;Vu et al 2016;). To reduce background activity, all assay reagents were dissolved in water; PES and DCPIP solutions were prepared in opaque tubes and kept on ice; and cell-free extracts were preincubated for 2 minutes at 30 °C as recommended (Jahn et al 2020).…”