Suprasil quartz epr tubes

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)].

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)}.

EPR spectra were recorded using a Bruker ELEXYSYS‐E500/E580 X‐band EPR spectrometer (Bruker GmbH, Rheinstetten, Germany). The microwave power was set to 30 dB, the modulation amplitude set to 5 G, a time constant of 41 ms, a conversion time of 41 ms, a sweep time of 84 s, the receiver gain set to 60 dB and an average microwave frequency of 9.384 GHz. Throughout the measurements, a temperature of 20 K was maintained via an Oxford Instruments ESR900 helium flow cryostat coupled to an ITC503 controller from the same manufacturer. EPR experiments were carried out at 20 K and employed 0.2 mW microwave power, 100 kHz modulation frequency and 5 G (0.5 mT) modulation amplitude. All EPR samples were prepared in an anaerobic glove box (O₂ < 2 ppm) and placed in 4‐mm Suprasil Quartz EPR Tubes (Wilmad‐LabGlass, NJ, USA). Tubes were sealed with a Suba‐Seal rubber stopper under anaerobic conditions and immediately frozen in liquid N₂ to prevent reoxidation.

Samples of 100 μM NpRdhA altered proteins were prepared as isolated or reduced and transferred in volumes of 300 μL into 4 mm Suprasil quartz EPR tubes (Wilmad, Vineland, NJ, USA) under anaerobic conditions. The tubes were anaerobically sealed, frozen and stored in liquid nitrogen. Experimental parameters were as follows: microwave power 0.5 mW, modulation frequency 100 kHz, modulation amplitude 5 G, temperature 30 K. Spectra were obtained using a Bruker ELEXSYS E500 spectrometer (Coventry, UK), Super high Q resonator (ER4122SHQ), Oxford Instruments (Abingdon, UK) ESR900 cryostat and ITC503 temperature controller. Spectra were sums of between 8 and 16 scans.

The reduction potentials of the T1 and T2Cu centers in Br^2DNiR were determined by titration with sodium dithionite. To facilitate communication between the electrode and the protein, redox mediators were used. The electrochemical potential of the solution was measured using a Thermo Orion oxygen reduction potential electrode at 25 °C. A factor of +207 mV was used to correct values to the standard hydrogen electrode. During titration against dithionite, samples were withdrawn for electron paramagnetic resonance (EPR) analysis. They were placed in 4-mm OD Suprasil quartz EPR tubes (Wilmad LabGlass) under anaerobic conditions and immediately frozen in liquid N₂. To determine the redox potentials of the two copper centers, EPR spectroscopy was used. Continuous wave X-band EPR spectra (∼9.4 GHz) were recorded using a Bruker ELEXSYS E580 EPR spectrometer (Bruker GmbH) using a super-high-Q resonator. Temperature was maintained using an Oxford Instruments ESR900 helium flow cryostat coupled to an ITC 503 controller from the same manufacturer. EPR experiments were carried out at 20 K and employed 0.5-mW microwave power, 100-kHz modulation frequency, and 5-G (0.5 mT) modulation amplitude. Redox potentials of the copper centers were determined by fitting data to the Nernst equation.

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)}