Suprasil quartz epr tube

All EPR samples were prepared in Tris-HCl buffer pH 7.5. Samples containing ~300 μM of wild-type TtCBD and TtCBD-H132A were transferred into 4 mm outer diameter/3 mm inner diameter Suprasil quartz EPR tubes (Wilmad LabGlass) and frozen in liquid N₂. The photoactivation of the TtCBD and TtCBD-H132A was carried out at room temperature under anaerobic conditions. Optical irradiation at 530 nm (500 mW) was accomplished (for the specified duration described in the text/legend) using a Thorlabs Mounted High Power LED (M530L3) with the output beam collimated using a Thorlabs collimation adaptor (SM2F32-A). All EPR samples were measured on a Bruker EMXplus EPR spectrometer equipped with a Bruker ER 4112SHQ X-band resonator. Sample cooling was achieved using a Bruker Stinger^{34 (link)} cryogen-free system mated to an Oxford Instruments ESR900 cryostat, and temperature was controlled using an Oxford Instruments MercuryITC. The optimum conditions used for recording the spectra are given below; microwave power 30 dB (0.22 mW), modulation amplitude 5 G, time constant 82 ms, conversion time 25 ms, sweep time 90 s, receiver gain 30 dB and an average microwave frequency of 9.383 GHz. All EPR spectra were measured as a frozen solution at 20 K, respectively. The analysis of the continuous wave EPR spectra were performed using EasySpin toolbox (5.2.28) for the Matlab program package^{35 (link)}.

All solvents
were of analytic grade and were purchased from Sigma-Aldrich (Dorset,
UK). All EPR samples were prepared with either neat DMSO (5 mM; final
concentration of Cu(II) complex) or 7:1 abs. ethanol: DMSO solvent
mixture (625 μM; final concentration of Cu(II) complex) in an
aerobic condition. Samples containing ∼5 mM/625 μM of
Cu(II) complexes and polycrystalline powders of samples were transferred
into 4 mm outer diameter/3 mm inner diameter Suprasil quartz
EPR tubes (Wilmad LabGlass) and frozen in liquid N₂. All
EPR samples were measured on a Bruker EMXplus EPR spectrometer equipped
with a Bruker ER 4112SHQ X-band resonator. Sample cooling was achieved
using a Bruker Stinger cryogen free system mated to an Oxford Instruments
ESR900 cryostat, temperature control was maintained using an Oxford
Instruments MercuryITC.^{38 (link)−40 (link)} The optimum conditions used for
recording the spectra are given below: microwave power 30 dB (0.219
mW), modulation amplitude 5 G, time constant 82 ms, conversion time
16.67 ms, sweep time 60 s, receiver gain 30 dB, and an average microwave
frequency of 9.368 GHz. All EPR spectra were measured as frozen solutions
at 20 K, respectively. The analysis of the continuous wave EPR spectra
and simulations were performed using EasySpin toolbox (5.2.35) for
the Matlab (MATLAB_R2022a) program package.^{35 (link)}

All EPR samples were prepared in a 10 mm HPEPS buffer with 250 mm NaCl, 0.5 mm TCEP and 10% glycerol (v/v) (pH 7.6) in an aerobic condition. Samples containing ~ 200 μmAbCntA, 75 mm nicotinamide adenine dinucleotide (NADH) were transferred into 4‐mm Suprasil quartz EPR tubes (Wilmad LabGlass, Vineland, NJ, USA) and frozen in liquid N₂. Annealing of the samples was performed at room temperature for the specified time‐duration in the figure caption. All EPR samples were measured on a Bruker EMX‐Plus EPR spectrometer (Coventry, UK) equipped with a Bruker ER 4112SHQ X‐band resonator as reported previously [39 (link)]. Sample cooling was achieved using a Bruker Stinger [40 (link)] cryogen‐free system mated to an Oxford Instruments ESR900 cryostat, and temperature was controlled using an Oxford Instruments MercuryITC (Abingdon, UK). The optimum conditions used for recording the spectra are given below; microwave power 30 dB (0.2 mW), modulation amplitude 5 G, time constant 82 ms, conversion time 12 ms, sweep time 120 s, receiver gain 30 dB and an average microwave frequency of 9.383 GHz, temperature 20 K. cw‐EPR spectra for the AbCntA‐WT, single (C206A and C209A) and double (C206AC209A) mutants of CntA were recorded in the presence and absence of CntB + NADH + carnitine, as reported previously [18 (link), 41 (link)].