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. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.
Coupled assay; DCPIP; EPR spectroscopy; Electron acceptors; Enzymatic assay; Methanol dehydrogenase; PES; PMS; UV–Vis spectroscopy; Wurster’s blue.
Conflict of interest statement
All authors declare that they have no conflict of interest.
Electron acceptors and dyes that have been used to assess MDH activity (their degradation products are also shown): PMS (
1a), phenazine ( 1b) and its oxidation product pyocyanin (PMS ox, 1c). PES, ( 2a) and its oxidation product (PES ox, 2b). DCPIP, ( 3), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD) dihydrochloride (TMPDD, 4a) and N, N, N ′, N′-tetramethyl- p-phenylenediamine perchlorate (Wurster’s blue, WB, 4b)
Upon two electron reduction, DCPIP undergoes a distinct color change. Usually the sodium salt and neutral to alkaline pH are employed in MDH assays. Hence, one of the deprotonated forms is shown
Absorbance spectra of 50 µM DCPIP (
3) and 100 µM WB ( 4b) in 100 mM multicomponent buffer of pH 6 (for DCPIP), pH 7 or pH 9. Fresh samples were prepared by diluting a 2 mM stock solution of the dye with the corresponding buffer. Spectra were collected at a Cary60 UV–Vis spectrophotometer at room temperature and corrected for the buffer baseline
A selection of buffers that have been used in MDH assays. The piperazine ring in PIPES and HEPES shown in red may cause problems when investigating redox reactions. The amine of Tris can react with formaldehyde, a substrate/product of many enzymes including MDH. Buffers shown in blue are known to complex or precipitate lanthanides and may thus compete with the enzyme for the metal ion in the active site
PMS was exposed to different conditions and the product mixture was analyzed using mass spectrometry. Structures and exact masses of the cations of PMS, phenazine (as its protonated derivative), and pyocyanin (as its protonated derivative). Products of the reaction of PMS with ammonia and cyanide, according to the literature [61, 62], and a proposed structure of ethyl-pyocyanin, a decomposition product of PES, are also shown (for more details see Supporting Information)
EPR spectra of 10 mM PMS (
a) and PES ( b) in MilliQ water (pH 6) or 100 mM multicomponent (MC) buffer pH 7.2 or pH 9. Solutions were prepared on a cloudy day and were either stored in an amber tube at 4 °C (black line), at RT in the dark (green line) or heated at 45 °C for 15 min in an amber tube (blue line). Additional samples were exposed to either daylight (orange line) or UV light of 254 nm (pink line) for 5 min each. Spectra were recorded at room temperature using an EMXnano EPR spectrometer
EPR spectra of 10 mM PMS (
a) and PES ( b) in MilliQ water (pH 6, purple line); 20 mM PIPES buffer of pH 6.2 (red line) and pH 7.2 (blue line); 20 mM potassium phosphate buffer of pH 7.2 (grey line). Solutions were prepared on a sunny day and were exposed to daylight for 5 min. Spectra were recorded at RT using an EMXnano EPR spectrometer
Specific activity (SA, in μmol min
−1 mg −1) of MDH using different PMS and PES batches of different purities and suppliers. M. extorquens AM1 La-MDH (untagged, 100 nM) in multicomponent buffer (100 mM, pH 9), 15 mM NH 4Cl at 30 °C. All samples contained 100 μM DCPIP and 50 mM MeOH, with 1 mM PES or PMS. Total volume in all wells was 200 μL. The reaction was monitored at 600 nm. SA1 and SA3 were determined by a different pair of hands than SA2 and are technical replicates
The radical cation Wurster’s blue (
4b) can undergo reduction to TMPD ( 4a) and can be used to monitor MDH activity
UV–Vis and EPR spectra of 200 µM WB in solution over time. 2 mM WB samples in MilliQ water (
a, b) or 100 mM multicomponent buffer pH 9 ( c) were stored on ice. In the case of a WB was diluted with MilliQ water. Samples of b and c were diluted in 100 mM multicomponent buffer, pH 9. UV–Vis spectra of triplicates ( a, c) and duplicates ( b) were recorded at 30 °C on an Epoch2 spectrophotometer without path length correction. MilliQ water and buffer baselines were subtracted from the corresponding spectra. The standard deviation was less than 7%. EPR spectra were recorded on an EMXnano EPR spectrometer at room temperature and in the dark. Blue line: fresh sample, red line: sample that has been stored on ice for 3 h in amber tubes
UV–Vis spectra of differently stored WB in 100 mM multicomponent buffer pH 9. 2 mM WB samples were stored in MilliQ water and diluted with buffer to a concentration of 200 µM before measurement. Spectra of triplicates were recorded at 30 °C on an Epoch2 plate reader without path length correction. The buffer baseline was subtracted from the spectrum. The standard deviation was less than 10%. (FF, flash frozen)
pH dependence of WB in different buffers. Conditions were as follows: 200 µM WB in 20 mM buffer of different pH, heated for 1 h at 30 °C. Absorbance at 610 nm was monitored with an Epoch2 plate reader. Experimental and technical (CHES and CAPS) triplicates with standard deviations are shown. Data were path length corrected to 1 cm
WB dependence of AM1 La-MDH and SolV Eu-MDH. The specific activity (SA, in μmol min
−1 mg −1) of His-tagged AM1 La-MDH (left) was determined in 100 mM multicomponent buffer, pH 9, with 15 mM NH 4Cl. SolV Eu-MDH activity (right) was measured in 100 mM multicomponent buffer, pH 7.2, with added 20 µM Eu(III). The WB concentration was varied, and protein concentration was constant at 100 nM for AM1 La-MDH and 200 nM for SolV Eu-MDH. The assay was performed with 50 mM MeOH at 30 °C and 610 nm. The total volume in wells was 200 µL. All SA are technical replicates. SA1 and SA2 were determined by different pairs of hands than SA3. Data were collected at an Epoch2 plate reader
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